(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Children's Library | Biodiversity Heritage Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

Full text of "Characterization of the Chediak-Higashi syndrome gene in human and mouse"

CHARACTERIZATION OF THE CHEDIAK-HIGASHI SYNDROME GENE IN 

HUMAN AND MOUSE 



By 

VELIZAR T. TCHERNEV 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1998 



Copyright 1998 

by 

Velizar T. Tchernev 



To my parents 



ACKNOWLEDGMENTS 

The author would like to thank his mentor, Dr. Stephen F. Kingsmore, and the 
supervisory committee members, Dr. M. Wallace, Dr. E. Wakeland, Dr. E. Sobel and Dr. 
M. Bubb, for their valuable advice and encouragement. 

The author's appreciation is extended to all former and present members of Dr. 
Kingsmore's laboratory, Dr. Maria D.F.S. Barbosa, Dr. Vishnu S. Mishra, John C. Detter, 
Dr. Elizabeth B. McMurtrie, Quan A. Nguyen, Sandra M. Holt, Juan M. Teodoro and 
Andrea Hofig, as well as to Dr. Krishnan Nandabalan and Dr. Madan Kumar from 
CuraGen Corporation, who contributed to the successful completion of this work. 

The author wishes to express his gratitude to Ms. Anna V. Gueorguieva and Mr. 
Nathan S. Collier for their kindness and continuous support. 

Finally, but most importantly, the author would like to thank his parents Lilia 
Jeleva and Tzvetan Tchernev for their loving care and guidance, his beloved wife Ralitza 
Gueorguieva for her invaluable support and inspiration, and his fellow-players Ivajlo 
Kortezov, Rada Kortezova, Ludwig Lubih, Anastas Assenov and Isak Beraha for their 
true friendship and thought-provoking conversations. 



IV 



TABLE OF CONTENTS 

page 

ACKNOWLEDGMENTS iv 

ABSTRACT vii 

INTRODUCTION 1 

MATERIALS AND METHODS 22 

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 

Amplification of CHS Patient and Beige Mouse cDNAs 22 

TA Cloning of PCR Products and Plasmid Purification 23 

Single-Strand Conformation Polymorphism (SSCP) Analysis 24 

DNA Sequencing and Sequence Analysis 24 

Allele-Specific Oligonucleotide Analysis 25 

PCR Amplification of Mouse Lyst cDNAIsoforms 25 

Cloning of Human ZESTcDNAs 26 

Molecular Probes 28 

Isolation of LYST2 cDNA Clones 29 

Southern Blot Analysis and Autoradiography 30 

Northern Blot Analysis 30 

Simple Sequence Length Polymorphism (SSLP) PCR Amplification 31 

Backcross Mouse Panel and Genetic Mapping 31 

Cloning of LYST and LYST2 Bait cDNA Fragments in Yeast Two- 
Hybrid Vectors 32 

Yeast Two-Hybrid Screens 36 

Confirmation of the Specificity of Protein Interactions by the Yeast 

Two-Hybrid System 40 

RESULTS 43 

Identification of Mutations in Patients with Chediak-Higashi 

Syndrome and in Beige Mice 43 

Identification and Characterization of Lyst mRNA Isoforms 52 

Identification and Characterization oiLyst2, a Brain-Specific Member 

of the Chediak-Higashi Syndrome Gene Family 61 

Identification and Sequence Analysis 61 

Genetic Mapping 62 

Expression Analysis 67 



Identification of Proteins that Interact with the CHS Protein and with 

LYST2, Using a Yeast Two-Hybrid Approach 71 

IP-1 (EST cg50136.f6, APPENDIX, Sequence 13) 76 

IP-2 (EST AA010799, APPENDIX, Sequence 14) 77 

IP-3 (EST cg50136.cl0, APPENDIX, Sequence 15) 77 

IP-4 (EST cg50136.a7, APPENDIX, Sequenced) 78 

IP-5 (EST cg50175.c7, APPENDIX, Sequence 17) 79 

IP-6 (EST cg50138.g5, APPENDIX, Sequence 18) 80 

IP-7 (EST cg50173.dl0, APPENDIX, Sequence 19) 81 

IP-8 (EST cg50175.h7, APPENDIX, Sequence 20) 82 

IP-9 (EST KIAA0192, APPENDIX, Sequence 21) 83 

IP-10 (EST cg50136.a5.b, APPENDIX, Sequence 22) 83 

IP-11 (EST cg51287.dl0, APPENDIX, Sequence 23) 84 

IP-12 (EST cg49432.h3.b, APPENDIX, Sequence 24) 85 

IP-1 3 (EST cg50175.cl 1, APPENDIX, Sequence 25) 86 

DISCUSSION 88 

Identification and Characterization of Lyst mRNA Isoforms 88 

Identification of Mutations in Patients with Chediak-Higashi 

Syndrome and in Beige Mice 92 

Identification and Characterization of Lystl, a Brain-Specific Member 

of the Chediak-Higashi Syndrome Gene Family 94 

Identification of Proteins that Interact with the CHS Protein, Using a 

Yeast Two-Hybrid Approach 98 

APPENDIX SEQUENCES OF MOLECULES INTERACTING WITH LYST 

ANDLYST2 123 

BIBLIOGRAPHY 177 

BIOGRAPHICAL SKETCH 192 



VI 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfillment of the 

Requirements for the Degree of Doctor of Philosophy 

CHARACTERIZATION OF THE CHEDIAK-HIGASHI SYNDROME GENE IN 

HUMAN AND MOUSE 

By 

Velizar T. Tchernev 

December 1998 

Chairman: Stephen F. Kingsmore 

Major Department: Pathology, Immunology and Laboratory Medicine 

Chediak-Higashi syndrome (CHS) is an autosomal recessive, immune deficiency 

disorder of human and mouse (beige, bg) that is characterized by abnormal intracellular 

protein transport to and from the lysosome/late endosome. Recent reports have described 

the positional cloning of homologous genes, LYST and Lyst, that are mutated in human 

CHS and in bg mice, respectively. However, since the encoded proteins were novel and 

unlike any of the molecules previously implicated in vesicular trafficking, their 

identification did not prove immediately helpful in establishing the precise mechanism 

whereby CHS dysregulates protein transport. Therefore, the main goal of this dissertation 

was to characterize the Chediak-Higashi gene and its products in more detail using 

functional genomics approaches. We demonstrated that each of the previously reported 

bg gene sequences are derived from a single gene with alternatively spliced mRNAs. 

Alternative splicing results in bg gene isoforms (Lyst-l and Lyst-IT) that contain different 



vn 



3' regions. Similarly to mouse, the human mRNA isoforms arise from incomplete 
splicing and retention of a transcribed intron that encodes the C-terminus of the predicted 
LYST protein. Additional splicing complexity of smaller isoforms exists. The Lyst-lll 
isoform lacks exons a and p\ while in Lyst-YV, exons a, P and y are absent. Novel 
mutations were identified within the coding domain of LYST in several CHS patients and 
bg alleles. Interestingly, all bg and LYST mutations identified to date are predicted to 
result in either truncated or absent proteins. Mutation and expression analyses suggested 
that defects in the full-length mRNA alone can elicit Chediak-Higashi syndrome and that 
expression of the smaller isoform alone cannot compensate for loss of the largest isoform. 
We have also identified a novel gene in human (LYST2) and mouse (Lyst2) that appears 
to be a relative of the CHS gene, based on sequence similarity, predicted protein structure 
and on similar transcript size. Comparison of the relative abundance of LYST2 and LYST 
mRNAs suggests that LYST2 is expressed abundantly only in brain and therefore it may 
represent a brain-specific member of the CHS gene family. Using a modified yeast two- 
hybrid system, a human cDNA library was screened with baits from the coding domains 
of ZTSTand LYST2. Several proteins, which play important roles in protein transport and 
signal transduction, such as 14-3-3, casein kinase II, calmodulin and Hrs, were found to 
interact with LYST and/or LYST2. Many of the interacting proteins could be linked in a 
common pathway that regulates vesicular trafficking and degranulation. 



vin 



INTRODUCTION 



Chediak-Higashi syndrome (CHS) is a primary immunodeficiency, characterized 
by severe recurrent infections (Blume & Wolff, 1972), partial albinism (Windhorst et al, 
1968), nervous system abnormalities (Misra et al, 1991), high incidence of malignancy 
(Hayakawa et al, 1986) and predisposition to bleeding (Meyers et al, 1974). Eighty-five 
percent of patients undergo an accelerated lymphoma-like phase, characterized by fever, 
hepatosplenomegaly, lymphadenopathy, pancytopenia, coagulopathy and widespread 
lymphohistiocytic organ infiltrates. This complication is fatal in the absence of bone 
marrow transplantation. 

The first 3 cases of Chediak-Higashi syndrome were reported by Beguez-Cesar in 
1943, then one case by Steinbrink in 1948, and one case by Higashi in 1954. Chediak 
published certain aspects of Beguez-Cesar' s cases in 1952, and Sato in 1955 recognized 
the similarities between Chediak' s and Higashi 's data, inventing the eponym "Chediak- 
Higashi syndrome." Except for the occasional usage of single names or combinations of 
the above authors' names, the disease has been most commonly referred to as the 
Chediak-Higashi syndrome. 

Approximately 1 50 cases of CHS have been reported in the literature. Males and 
females are affected equally. The mean age of death of CHS patients is 6 years. The 
Chediak-Higashi syndrome occurs with a low frequency throughout the world. Europe, 

1 



America and Japan are the areas from which the majority of the known cases have been 
described. While most of the reports deal with isolated cases, a unique cluster of Chediak- 
Higashi syndrome patients has also been published (Ramirez-Duque et al, 1982). 
Fourteen cases of CHS were found in 12 families living in a defined and relatively 
isolated geographical area (Pregonero) in the Venezuelan Andes. The patients were pre- 
school children except one 1 1 -year-old female. Six of the patients were male. All showed 
the same typical clinical characteristics of the syndrome. Since CHS is an autosomal 
recessive disease (Sato, 1955), the existence of this high-frequency cluster of CHS 
patients in a restricted region could be explained by a founder effect in an isolated 
population with inbreeding. 

The presence of giant, perinuclear vesicles with protein markers characteristic of 
late endosomes and mature lysosomes is pathognomonic for CHS (Burkhardt et al, 
1993). The organelles are abnormal both in size (enlarged) and in cytoplasmic 
distribution (perinuclear). While the late endosomal/lysosomal compartment is uniformly 
affected in CHS, the histological designation of affected organelles varies by cell type: 
melanosomes are affected in CHS melanocytes, while secretory granules are affected in 
CHS neutrophils, eosinophils, mast cells, cytolytic T-cells, proximal renal tubules, and 
lysosomes are affected in monocytes and fibroblasts. The giant organelles are thought to 
result from dysregulated fusion of primary endosomes and lysosomes (Burkhardt et al., 
1993; Willingham et al, 1981). The morphological abnormalities of bg and CHS 
lysosomes are accompanied by compartmental mis-sorting of proteins such as elastase, (3- 
glucuronidase, and cathepsin G (Holcombe et al., 1994). 



The disease also occurs in other mammalian species - mouse, rat, cat, cattle, 
mink, fox and killer whale. Beige (bg) is a well described mouse model of CHS. The 
clinical and pathological features of CHS patients and bg mice are indistinguishable. 
Prior to the cloning of bg, the strongest evidence of homologous defects in beige and 
CHS came from interspecific genetic complementation studies, which demonstrated that 
fusion of bg and CHS fibroblasts failed to reverse lysosomal abnormalities, while fusion 
with normal cells did eliminate cytological defects of bg or CHS fibroblasts (Perou and 
Kaplan, 1993; Penner and Prieur, 1987). 

Partial albinism is one of the main clinical manifestations of the Chediak-Higashi 
syndrome. Most patients have hypopigmentation of their hair, skin and ocular fundi, as 
well as misrouting of the optic nerve fibers that is characteristic of all types of albinism. 
Windhorst et ai, 1966, 1968 reported the presence of giant melanosomes in melanocytes 
from CHS patients. Some melanosomes were highly irregular, with a suggestion of fusion 
of separate granules. Most epidermal melanosomes from a CHS patient, observed by 
Zhao et a}., 1994, were several times larger than normal, relatively amelanotic, and 
occasionally clustered into complexes. No melanosomes were detected in keratinocytes 
adjacent to melanocytes, suggesting that the normal transport of melanosomes from 
melanocytes to neighboring keratinocytes is defective. After DOPA histochemistry, used 
to localize functional tyrosinase, reaction product was observed throughout the 
cytoplasm, but not in most of the aberrant large melanosomes and complexes, indicating 
that functional tyrosinase is either not translocated to or is not retained within the giant 
granules. In contrast to control melanocytes, DOPA-positive vesicles migrated far beyond 
their usual perinuclear and trans Golgi network location and were seen in the vicinity of 



the plasma membrane in melanocytes from CHS patients. When several melanocyte- 
specific proteins were localized histochemically or immunohistochemically, they were 
predominantly detected in the perinuclear area and less prevalent in the dendrites of CHS 
cells, compared to their relatively uniform distribution throughout the cytoplasm of 
control cells (Zhao et ai, 1994), suggesting an inefficient intracellular translocation of 
these proteins. Medium, conditioned by CHS cells for 1 to 3 days, displayed increasing 
amounts of tyrosinase activity that did not exist in medium conditioned by numerous 
control cell lines, ^-glucuronidase was also released into the culture medium by CHS 
melanocytes but not by control melanocytes. These data suggest that the low tyrosinase 
activity in giant melanosomes results from mislocalization and eventual extracellular 
secretion of tyrosinase. This hypothesis is supported by the presence of DOPA-positive 
vesicles beneath the plasma membrane where fusion/secretion could occur. 

Patients with Chediak-Higashi syndrome, especially those who survive into 
adulthood, and elderly beige mice, develop serious neurologic defects. Lockman et ah, 
1967, reported a patient with the typical clinical and cytological picture of CHS and a 
severe motor neuropathy. A median nerve biopsy showed that many Schwann cells 
contained large cytoplasmic particles which the authors considered to be abnormal 
lysosomes. Two cases with lymphohistiocytic infiltration of the sural and femoral nerves 
were described by Sung et ai, 1969. The myelin sheaths and axons showed swelling and 
fragmentation or had disappeared. Schwann cells were hypertrophic and contained 
cytoplasmic inclusions. Ultrastructural examination of dorsal root and sympathetic 
ganglion cells demonstrated inclusions resembling lysosomes and lipofuscin granules. 



Pettit and Berdal, 1984, reported a 25-year-old man with a neurologic disorder that 
resembled a spinocerebellar degeneration and Parkinsonism. Cerebellar cortical atrophy 
(Kondo et al., 1994) and diffuse atrophy of the brain with abnormalities in the 
periventricular and corona radiata regions were detected by computer tomography and 
magnetic resonance imaging (Ballard et al, 1994). The oldest known CHS patient first 
seen with a neurologic disorder in early adult life was described by Uyama et al, 1994. 
This 39-year-old woman developed mental deterioration, Parkinsonism, muscular atrophy 
of limbs, and loss of tendon reflexes. MRI showed marked temporal dominant brain 
atrophy and diffuse spinal cord atrophy. Similarly, beige mice that survive to 1 7 months 
of age show a progressive neurologic disorder accompanied by nearly complete loss of 
cerebellar Purkinje cells (Murphy and Roths, 1978). Guo et al, 1992, reported 
cytoarchitectonic abnormalities in the brains of beige mice. In the cerebellum, Purkinje 
cells and clusters of granule cells, and occasionally glia cells, were located ectopically in 
the molecular level. In the hippocampus, ectopically situated pyramidal cells were found. 
The infrapyramidal mossy fiber layer was not compact, but appeared as clumps of 
scattered fiber bundles. Pezeshkpour et al, 1986, reported the presence of giant 
lysosomes in Schwann cells in a nerve biopsy from a CHS patient. Peripheral neuropathy 
in a 3 3 -year-old brother and a 29-year-old sister with CHS were described by Misra et al, 
1991. Both showed evidence of a sensory neuropathy associated with central nervous 
system involvement. Nerve conduction studies indicated an axonal neuropathy. Sural 
nerve biopsy demonstrated a loss of myelinated nerve fibers, particularly those of larger 
size, and of unmyelilnated axons. By light and electron microscopy, granulated cells with 
the staining properties of mast cells were present in the endoneurium. Instead of being the 



usual 0.1-0.2 urn in diameter, the granules were approximately 10 times larger and with 
unusual appearance. Blume and Wolf, 1972, reported irregular giant inclusions, thought 
to be lysosomes, in the cytoplasm of Schwann cells. These morphologic observations of 
abnormally large granules suggest that the neurological aberrations in CHS result from 
defective vesicular transport of molecules that are important for the normal cellular 
function. 

Bleeding tendency is another major clinical manifestation in patients and animals 
with CHS. While the platelet numbers are normal (Novak et al, 1985; Phillips et al, 
1967), dense platelet granules are virtually absent, whole-blood serotonin is markedly 
decreased, and platelets have reduced ability to accumulate serotonin (Meyers et al., 
1974; Meyers et al, 1983). The prolonged bleeding time and low serotonin 
concentrations were converted to normal values following transplantation of normal bone 
marrow in beige mice. Likewise, control mice displayed symptoms of storage pool 
deficiency when transplanted with mutant marrow (Novak et al., 1985). These studies 
demonstrate that the CHS platelet storage pool deficiency results from a defect in bone 
marrow precursor cells. In agreement with this conclusion, Menard and Meyers, 1988, 
showed that the dense granule precursors in maturing and mature megakaryocytes were 
absent. Abnormal platelet aggregation was described in 6 patents with CHS (Apitz-Castro 
et al., 1985). The total content and the maximal amounts of the dense granule constituents 
secreted after thrombin stimulation were greatly decreased. CHS platelets loaded with 
radiolabeled serotonin showed a spontaneous release of radioactivity not observed in 
normal platelets under the same conditions. Similar defects were reported in cats (Colgan 



et al, 1989) and in blue foxes (Sjaastad et al, 1990) with Chediak-Higashi syndrome. In 
beige mice, platelet aggregation after stimulation was significantly decreased compared 
to that of normal mice (Pratt et al, 1991). Platelets from beige mice were approximately 
10 times more sensitive to prostacyclin inhibition of collagen-induced aggregation than 
control platelets. The stores of serotonin and adenine nucleotides were decreased. Rendu 
et al, 1983, reported a large reduction in the number of serotonin-storage granules (dense 
bodies) but otherwise normal ultrastructure and normal amounts of a- and catalase- 
positive granules in 3 patients with Chediak-Higashi syndrome. The platelet release 
reaction, studied with thrombin as an inducer, was impaired. The serotonin uptake by the 
patients' platelets was low and its metabolism was increased. These findings show that 
human CHS platelets are deficient in the storage pool of dense granule substances and 
suggest that this granule defect influences the release mechanism of other granule 
constituents, leading to abnormal aggregation and bleeding predisposition. 

Most morbidity and mortality in CHS result from immune deficiency, with severe 
defects in innate immunity (granulocytes and NK cells) and relatively mild impairment of 
acquired immune function (B and T cells). 

The initial immune defect recognized in CHS was a striking reduction in the 
ability of granulocytes to kill phagocytosed microorganisms (Blume et al., 1968; Gallin 
et al., 1974). Phagocytosis of radiolabeled Staphylococcus aureus by CHS granulocytes 
was normal. However, there was reduced bactericidal activity of S. aureus and group D 
Streptococcus by CHS cells through 90 min of incubation. This defect was most 
pronounced at early time periods and was related to impaired intracellular killing. There 



8 

are occasional reports suggesting more widespread neutrophil abnormalities. In one study 
(Bellinati-Pires et al, 1992), in which phagocytosis and killing of Staphylococcus aureus 
were simultaneously evaluated by a fluorochrome phagocytosis assay, both appeared to 
be deficient in neutrophils form two patients with CHS. The presence of giant 
phagolysosomes, enclosing bacteria in active proliferation 45 min after phagocytosis was 
noteworthy, and corresponded with the impaired bactericidal activity of these leukocytes. 
In an under-agarose assay, phagocytes of homozygote and phenotypically normal 
heterozygote CHS cats recognized and responded equally well to bacterial stimuli as did 
cells from control animals but traveled shorter distances primarily because of a reduced 
inherent motility. Similar results were obtained when phagocyte chemotaxis was 
evaluated with zymosan-activated serum (Colgan et al., 1992). Mildly defective CHS 
neutrophil and monocyte locomotion was discovered by the micropore filter technique 
using modified Boyden chambers (Yegin et al, 1983). Isolated reports suggest that 
diminished surface expression of C3bi receptor and decreased oxygen radical generation 
by polymorphonuclear leukocytes (PMNs) might be some of the factors to explain the 
moderately defective PMN mobility, chemotaxis and bactericidal activity (Cairo et al., 
1988; Kubo et al., 1987). While the literature contains some conflicting data, the 
consensus is that, with the exception of the ability to kill phagocytosed microorganisms, 
other granulocyte activities, such as phagocytosis, chemotaxis and protein degradation, 
are relatively well preserved in CHS (Gallin et al, 1974; Clawson et al., 1978). 
Analogous defects occur in CHS monocytes and macrophages — cytolysis of tumor 
targets is impaired, while phagocytosis and chemotaxis are almost normal (Gallin et al., 
1975; Mahoney et al., 1980). CHS selectively involves crystalloid granules in late 



eosinophils, while the non-crystalloid granules are preserved, suggesting that the 
underlying pathology does not affect all lysosomal subpopulations (Hamanaka et al, 
1993). 

The second category of CHS immune defects is a virtual ablation of natural killer 
(NK) cell activity and antibody dependent cellular cytotoxicity (Roder and Duwe, 1979; 
Roder et al, 1979, 1982). Mature NK cells demonstrate low lytic efficiency, assessed by 
conventional 4 h chromium release assay and by a more recently developed single cell 
liquid cytotoxic assay. The NK cells were not activated by prolonged in vitro incubation 
with IFN-y, but they exhibited IL-2 induced cytotoxicity, though the magnitude of 
induction was uniformly less than in controls (Virelizier and Griscelli, 1980; Holcombe, 
1992). CHS NK cells have the necessary cellular structures required for their role as lytic 
effector cells, but lack cytotoxic function due to a relative refractoriness in initiating the 
post-binding lytic mechanism. NK cells in CHS patients are blocked at a post- 
recognition, post-activation step in the cytolytic pathway, subsequent to the burst of 
oxygen intermediates but preceding the lethal hit. In summary, while NK cells are present 
in normal numbers, bind target cells effectively and generate superoxide ions normally, 
exocytosis of lytic granules from the CHS NK cells is refractory (Targan and Oseas, 
1983; Roder et al., 1983; Brahmi, 1983). 

Tumor susceptibility of bg mice was examined as a direct test of the hypothesis 
that NK cells are involved in surveillance against neoplasia. Subcutaneous inoculation in 
a transplantation test yielded higher tumor take incidence in bg/bg mice than in 
heterozygous littermates. The differences were most striking in the early phase of the 



10 

observation period, with a majority of the total tumor takes in bg/bg registered within 2 
weeks, whereas only a small part of the tumors in bg/+ mice were palpable at this time. 
Thus, in addition to the increased incidence in homozygous mice, the progressively 
growing tumors appeared with a shorter latency than in controls. This was also reflected 
in the larger mean tumor diameter by 2 and 3 weeks and the earlier deaths among tumor- 
bearing bg/bg than bg/+ mice. These findings correlated well with the in vitro measured 
splenic NK activity against both tumors tested. Tumor cells had a low, but significant, 
sensitivity to bg/+ spleen cell cytotoxicity (5-10 % specific lysis), whereas bg/bg spleen 
consistently gave values below 3% specific lysis (Kaerre et al., 1980). In another study, a 
tumor line modified to be sensitive to NK cytotoxicity in vitro, demonstrated in vivo an 
increased growth rate, faster induction time and an enhanced metastatic capability in bg 
compared to control mice. This was not found with a tumor line insensitive to NK 
activity. In vivo activation of NK cells in bg and control mice resulted in decrease in 
tumor growth rate and metastatic frequency (Talmage et al., 1980). In a large Japanese 
cohort, non-Hodgkin's lymphoma occurred in over one third of CHS patients (Hayakawa 
et al., 1986). These results indicate that NK cells have an important function in the host's 
control of tumor growth and metastasis. The idea to use the bg model to dissect the 
contribution of NK cells to immunity, and in particular, to tumor immunosurveillance 
was dispelled somewhat, however, by the subsequent recognition of concomitant 
impairment, albeit mild, in T and B cell function. 

Although both B and T cells are affected in CHS, the abnormalities in the specific 
defense mechanisms are not as severe as those in natural immunity and have less 
importance for the pathogenesis of CHS. The pathognomonic giant cytoplasmic vesicles 



11 

are detected in B cells from CHS patients following activation and differentiation (Grossi 
et al, 1985). Moderate and variable reduction of LPS-induced B cell mitogenesis is 
demonstrated in CHS. The in vivo anti-trinitrophenyl antibody response to a thymus- 
independent antigen (TNP-Ficoll) was found to be significantly lower in C57BL/6J-6g 
than in C57BL/6J mice, although both strains responded similarly to an analogous 
thymus-dependent antigen (TNP-ovalbumin) (Pflumio et al, 1990). Since the marginal 
zone macrophages of the spleen were previously shown to be essential for the initiation of 
antibody responses to thymus-independent antigens, they might be another target of the 
bg mutation, possibly resulting in impaired antigen presentation to B cells. 

It is notable that homozygosity at the bg locus confers protection against immune 
complex glomerulonephritis to BXSB mice with systemic autoimmune disease (Clark et 
al., 1982). However, this phenomenon is probably not due to a decreased B cell function 
because the levels and types of autoantibodies remain unchanged in bg homozygotes. 

Extremely high antibody titers to the Epstein-Barr virus specific viral capsid 
antigen, to the restricted component of the EBV-induced early antigen complex and to the 
EBV-associated nuclear antigen were detected in CHS patients (Merino et al., 1983). It is 
likely that the elevated antibody titers reflect an increased production of these antigens 
due to defective NK and antibody-dependent cellular cytotoxicity (ADCC) activities. 

Cytotoxic lymphocytes (CTLs) from CHS patients are unable to destroy target 
cells recognized via the T cell receptor (TCR). Individual CTL clones show poor killing 
that can be increased in longer assays. However, in the presence of cycloheximide, the 
small amount of killing is abolished, indicating that killing arises from newly synthesized 
proteins, rather than from proteins stored in granules. It has been shown that the CHS 



12 

CTL clones express normal levels of the lytic proteins granzyme A, granzyme B and 
perforin, which are processed properly during biosynthesis and targeted correctly to giant 
lytic granules. Despite the difference in size, CHS and normal lytic granules are similar, 
both contain the lysosomal enzyme cathepsin D and granzyme A, and lack the mannose- 
6-phosphate receptor. However, unlike normal CTL clones, the CHS CTLs are unable to 
secrete their giant granules in which the lytic proteins are stored. After cross-linking the 
TCR, CHS CTL clones fail to secrete granzyme A, as assayed by both enzyme release 
and confocal microscopy (Baetz et ah, 1995). 

The proportion of gamma-delta T cells among peripheral blood mononuclear cells 
is significantly increased in CHS. The cellular machinery for lysis of target cells in vitro 
is present in CHS-derived gamma-delta T cell clones (Holcombe et ah, 1990). This is the 
first example of a specific immunodeficiency disorder with a relative expansion of these 
T cells. 

A short summary of the immunological features of CHS is presented in Table 1. 



13 



Table 1 . Main immunologic features in Chediak-Higashi syndrome 




INNATE IMMUNITY 


ACQUIRED IMMUNITY 


Granulocytes 


NK cells 


B cells 


T cells 


1 . Greatly reduced 


1 . Very low lytic 


1 . Reduced antibody 


1 . Decreased ability 


ability to kill 


activity caused by 


response to thymus- 


ofCTLs to kill target 


phagocytosed micro- 


defective exocytosis 


independent antigens 


cells, caused by 


organisms 


of lytic granules 


(may be due to 


defective secretion of 


2. Chemotaxis, 


2. Numbers of NK 


impaired natural 


lytic granules 


phagocytosis and 


cells, binding to tar- 


immunity) 


2. Relative expansion 


other activities are 


get cells, and oxygen 


2. Mildly reduced 


of gamma-delta T 


relatively well 


radical production are 


proliferative response 


cell population 


preserved 


normal 


to LPS 




PATHOGNOMONIC GIANT PERINUCLEAR VESICLES 



14 

Most mortality in CHS patients is caused by two manifestations of their immune 
deficiency — severe pyogenic infections and the accelerated phase. Lung, upper 
respiratory tract and skin infections caused by Staphylococcus, Streptococcus and 
Pneumococcus predominate in CHS, although a wide variety of infection sites and 
organisms occur. The accelerated phase is considered a clinicopathologic expression of a 
virus-associated lymphoproliferation because of low CD4 + to CD8 + T lymphocyte ratio, 
presence of Epstein-Barr virus genome in the mononuclear cells of the lymph node, blood 
and bone marrow, and possible clinical responses to acyclovir. The current treatment for 
CHS patients ranges from vitamins to cytostatic agents and bone marrow transplantation, 
but the prognosis remains poor because of increasing susceptibility to infections and 
progressive neurologic deterioration. Several studies suggest novel strategies for 
immunomodulation: recombinant canine granulocyte colony-stimulating factor to 
improve the neutrophil function (Colgan et ah, 1992), IL-2 to induce cytotoxicity of CHS 
lymphocytes (Holcombe, 1992), and high-dose intravenous gammaglobulin for 
management of the accelerated phase (Kinugawa and Ohtani, 1985). The goal of such 
therapies would be to partially compensate the immune cell dysfunction caused by the 
basic genetic defect and manifested as abnormal protein trafficking. 

The transport of newly synthesized proteins from the endoplasmic reticulum to 
the Golgi apparatus and from there to the cell surface and elsewhere, is mediated by 
transport vesicles, which transfer enclosed proteins by cycles of vesicle budding and 
fusion. The abnormalities in CHS can be explained by a compartmental mis-sorting of a 
variety of proteins, which are necessary for the proper function of the affected cell types, 
for example CHS CTLs cannot secrete stored granzyme A and other lytic proteins, and 



15 

therefore cannot lyse target cells efficiently. The morphologic manifestation of the 
protein sorting defect in CHS is the presence of giant perinuclear vesicles in the 
cytoplasm of the affected cells. The assortment of protein trafficking defects observed in 
CHS defies simple explanation: trafficking both to and from the late 
endosomal/lysosomal compartments is affected, and proteins which utilize both the 
cation-independent mannose-6-phosphate and other transport mechanisms are mis-sorted. 
In contrast, targeting of membrane proteins to lysosomal/late endosomal compartments 
(LAMP1, LAMP2, rab7, acid phosphatase) is intact. Experiments demonstrating 
spontaneous concavalin-A induced capping of CHS granulocytes suggest that the defect 
impairs interactions between microtubules and endosomes/lysosomes (Oliver et ah, 
1975). However, detailed examination of microtubules and microtubule-based motors has 
not disclosed an abnormality (Perou and Kaplan, 1993). In summary, abnormalities in 
lysosomal and endosomal processing may explain the clinical features of CHS, 
suggesting that the defect involves a protein that regulates certain aspects of trafficking to 
and from lysosomal/late endosomal compartments in many cell types. 

A summary of the mechanisms involved in the pathogenesis of Chediak-Higashi 
syndrome is presented in Fig.l. All clinical manifestations are caused by a common 
defect in the regulation of vesicular trafficking in different cell types: melanosomes are 
affected in melanocytes, resulting in partial albinism; dense granules in platelets are 
defective, leading to bleeding predisposition; lysosomes and secretory vesicles in 
numerous immune cells are deficient, causing frequent infections and increased incidence 
of tumors; and giant inclusions are present in Schwann and Purkinje cells, which could be 
associated with neurological abnormalities. 



16 



t 

UJ 

u- 

UJ 



o 

UJ 

I 

u 

I— I 
CO 

< 



-» 



co 

u 

I — 

CO 






C 

u 



-J 

< 
C 

CO 

O 
Q 

g 



t 



CO 

O 

CO 



-> 



-» 



-* 



-> 



-> 



CO 

- 

U 

— 

CO 

u. 
> 
> 

C 

U 
UJ 
CO 



->• 



O -> 

CO 



-> 



-> 



-» 



CO 

-J 

UJ 
U 



< 

u 

CO 



CO 
UJ 

-J 
UJ 
H 
< 
-J 



CO 

I 

U 

o 



-> 



-> 



CO 

-J 

1— 1 


CO 




I 


J 


CO 


0. 


UJ 


J 





U 


J 


CO 

c 


H 

CO 

< 


UJ 
U 

c^ 


UJ 


2 

GO 


z 




UJ 


CO 


CO 


a 


J 


UJ 

F 


< 


1— H 

S 


u 

C 

z 

o 


0H 

c 

u 

< 


O 

P 

UJ 

z 



CO 

UJ 

u 
o 

3C 
cu 

>- 



-> 



-» 



-> 



-J 

< 
u 

i— i 

o 

o 
-J 

o 

P 

UJ 

Z 





z 




o 





H 


5 


CO 


Q 





UJ 


cu 


UJ 


CO 




1— 1 

Q 






< 

p 

< 



S 



CO 

z 


UJ 

u 


o 


z 


H 


UJ 


U 


Q 


UJ 

u- 

5 


i— i 

z 


a 


cd 
O 


UJ 


2 


> 


P 


UJ 


H 


CO 




z 


Q 

UJ 
CO 


UJ 

p 


< 

UJ 


a 


Cd 



u 



2 

CO 

Z 

PQ 

-J 
< 



<u 



-u 



17 

The need for molecular understanding of the basic defect in Chediak-Higashi 
syndrome led to the first mapping studies, which were greatly facilitated by the existence 
of the mouse model beige. Linkage of satin (sa) with bg defined a new linkage group, 
later found to lie on proximal mouse chromosome (Chr) 13 (St. Amand and Cupp, 1958; 
Lyon and Meredith, 1969; Lane, 1971). Owen et al, 1986, showed that in mouse, bg is 
closely linked to the gene for the y-chain of the T cell receptor (TCR). However, in 3 
families with CHS, RFLPs in the TCR y-gene were inherited discordantly with CHS, 
demonstrating non-linkage (Holcombe et al, 1987) and suggesting a break in the region 
of homology between human chromosome 7p and mouse chromosome 13. Further 
refinement of the genetic map of proximal mouse Chr 13 (Justice et al, 1990) led to 
linkage of bg to the gene encoding nidogen (Nid), a structural basement membrane 
protein (Jenkins et al, 1991). No crossovers between Nid and bg were detected in 123 
mice, suggesting that the two loci are within approximately 2.4 cM. Since human nidogen 
{NID) has been assigned to Chr lq43 (Olsen et al, 1989), the close linkage between bg 
and Nid established a new group of homology between mouse Chr 13 and human Chr lq, 
suggesting that the gene responsible for the Chediak-Higashi syndrome is located in the 
telomeric region of human chromosome lq. These results confirmed the earlier 
observation of non-linkage between CHS and TCR-y (Holcombe et al, 1987) and the 
existence of a breaking point in the region of homology between mouse Chr 13 and 
human Chr 7p. Indeed, linkage between NID and CHS on human chromosome lq was 
reported by Goodrich and Holcombe in 1995. Homozygosity mapping using markers 
derived from distal human chromosome lq was carried out by Fukai et al., 1996, in four 



18 

inbred families or probands with typical childhood Chediak-Higashi syndrome. The 
human CHS gene was localized to an 18.8 cM interval, flanked by D1S446 and Dl SI 84, 
on Chr Iq42-q44. Barrat et al, 1996, studied 10 CHS patients from 9 families and 
mapped the CHS locus to an approximately 5 cM region between D1S163 and D1S2680 
on human chromosome Iq42.1-q42.2. As a first step in a positional cloning effort, 
Kingsmore et a/., 1996a, precisely localized the beige gene to a 0.24 cM interval on 
proximal mouse Chr 13 by segregation analysis. In a total of 726 mice from 3 different 
backcrosses, bg was found to cosegregate with 6 genetic markers (Nid Estm9, D13MU56, 
D13MU162, D13MU237 and D13MU240). The candidacy of Nid and Estm9 was 
evaluated by Southern, northern and RT-PCR analyses, which suggested that neither of 
these two genes represented the bg gene. However, interstrain differences in pulsed field 
restriction fragment lengths were observed, providing a useful and simple test for initial 
evaluation of candidate genes. A refined physical map of the bg critical region was 
reported by Kingsmore et al, 1996b. Interspersed repetitive element-polymerase chain 
reaction (IRE-PCR) and direct cDNA selection were used to identify 20 novel sequence 
tag sites which enabled the construction of 2 contigs composed of overlapping YAC and 
PI clones, covering approximately 2400 kb of the bg non-recombinant interval. In 1996, 
two groups simultaneously reported the identification of the homologous beige and 
Chediak-Higashi syndrome genes. Barbosa et al, 1996, isolated a gene designated Lyst 
(Lysosomal /rafficking regulator), from YAC 195A8 by direct selection with mouse 
spleen cDNA. It was shown that Lyst represented the beige gene with a complete open 
reading frame of 4635 nucleotides, encoding a protein of 1545 amino acids. It was also 
demonstrated that corresponding human clones with high degree of homology to mouse 



19 

Lyst represented the gene responsible for Chediak-Higashi syndrome. Perou et al, 1996b, 
also reported the identification of the beige gene by YAC complementation and 
positional cloning. Interestingly, the partial cDNA sequence isolated by Perou et al, did 
not match or even overlap with the sequence of Lyst, described by Barbosa et al. One of 
the aims of the present dissertation was to explain this discrepancy. This issue was 
resolved by the demonstration that each of the previously reported beige gene sequences 
was derived from a single gene by alternative splicing (Barbosa et al., 1997; Nagle et al., 
1996), as described in the RESULTS section of this dissertation. However, the 
expectations that the identification of the complete mouse and human cDNA sequences of 
the CHS gene would elucidate the precise function of the encoded protein, were not met. 
The 11403-bp full-length open reading frame corresponded to a polypeptide of 3,801 
amino acids, with an estimated molecular mass of 429,153 daltons. Although several 
putative domains were found in Lyst, the predicted protein was novel and unlike any of 
the molecules previously implicated in vesicular transport (Barbosa et al., 1996, 1997; 
Perou et al, 1996b; Nagle et al, 1996). A helical region of Lyst was similar to stathmin, 
a phosphoprotein that regulates the polymerization of microtubules and acts as a relay for 
intracellular signal transduction. Therefore, this region of Lyst potentially encodes a 
protein interaction domain that may regulate microtubule-mediated lysosome trafficking. 
Although the CHS protein (LYST) is not predicted to have transmembrane helices, the 
carboxy-terminal tetrapeptide (CYSP) of the mouse Lyst-II isoform is similar to known 
prenylation sites and may provide attachment to membranes through thioester linkage 
with the cysteine. A novel conserved domain, designated BEACH (BEige And CHs), was 
defined based on homology with open reading frames from S. cerevisiae, C. elegans and 



20 

a human cell division control protein 4-related protein (CDC4L). The function of the 
BEACH domain remains unknown. Helical regions in the CHS protein resemble ARM 
and HEAT repeat motifs, which tend to form long rods. Some HEAT-containing proteins 
are associated with vesicular trafficking but the functional significance of these motifs in 
Lyst is unclear. The C-terminal region of the CHS protein contains several consecutive 
WD40 motifs, which were originally identified in the P-subunit of the G-protein 
transducin. This WD40 region may form (3-sheets arranged in a propeller-like structure 
that is thought to mediate protein-protein interactions. Based on the presence of ARM, 
HEAT, BEACH and WD40 domains, it has been proposed that, similarly to the yeast 
serine/threonine protein kinase VPS 15, Lyst functions as a component of a membrane- 
associated signal transduction complex that regulates intracellular protein trafficking. The 
CHS protein contains multiple sites of potential phosphorylation by casein kinase II 
(CKII), PKC, cAMP-dependent protein kinase and tyrosine kinase. Lyst seems to contain 
helical bundles with clusters of phosphorylation sites at their ends. Phosphorylation of 
these positions could provide a control mechanism by causing conformational change in 
the bundles, thereby affecting interactions with other molecules. In summary, the 
domains identified in Lyst support its potential involvement in the regulation of signal 
transduction and protein trafficking but do not place the CHS protein in any known 
family and do not define any proteins that interact with Lyst, leaving the question about 
its mechanism of action unanswered. 

Therefore, the main objective of this dissertation was to characterize the Chediak- 
Higashi syndrome gene and its products in more detail, and to identify proteins that 



21 

interact with Lyst, in order to get some insights about its biological role. Following the 
cloning of the CHS gene in human and mouse, the following specific aims for the present 
dissertation were determined: 

1 . Identification of mutations in patients with Chediak-Higashi syndrome and in 
beige mice. 

2. Identification and characterization of Lyst mRNA isoforms. 

3. Identification and characterization of Lyst2, a brain-specific member of the 
Chediak-Higashi syndrome gene family. 

4. Identification of proteins that interact with the CHS protein and with LYST2, 
using a yeast two-hybrid approach. 

Detection of mutations in the CHS gene would help the identification of critical 
amino acids and domains in Lyst and would enable genotype-phenotype correlation, as 
well as the design of tests for early diagnosis of Chediak-Higashi syndrome. The 
characterization of different mRNA isoforms would determine the functional importance 
of individual alternatively spliced variants and their expression in different tissues. The 
identification of proteins that interact with Lyst would reveal the pathways in which the 
CHS protein is involved and would enhance our knowledge about the mechanisms of 
regulation of protein trafficking, particularly to and from the lysosomes. 



MATERIALS AND METHODS 



Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification of CHS 

Patient and Beige Mouse cDNAs 

Lymphoblastoid and fibroblast cell lines from CHS patients were obtained from 
the Coriell Institute for Medical Research (Camden, NJ). Mice carrying different alleles 
of the beige mutation and control mice were obtained from The Jackson Laboratory (Bar 
Harbor, ME), sacrificed, and their organs were harvested and homogenized. Total cellular 
RNA was isolated from human and mouse cells by extraction with phenol/guanidine 
isothiocyanate (TRIzol Reagent, Life Technologies Inc., Gaithersburg, MD). One to three 
ug of RNA were reverse transcribed using Superscript II RNase H" reverse transcriptase 
(Life Technologies Inc.), with oligo(dT)-primed first strand cDNA synthesis. DNA 
fragments of interest were PCR amplified with KlenTaq DNA polymerase (Clontech 
Laboratories, Palo Alto, CA) using the following amplification conditions: 94°C for 2 
min, then 30 cycles of 94°C for 20 s, 55°C for 15 s, 68°C for 1 min, followed by 6 min at 
68°C. PCR products were separated by electrophoresis on agarose gels, visualized by 
ethidium bromide staining and purified using Wizard PCR Preps DNA Purification 
System (Promega, Madison, WI). One pL of purified PCR product was used as template 
for a hemi-nested PCR amplification with KlenTaq DNA polymerase under the same 



22 



23 

conditions as above. PCR products were separated by electrophoresis on agarose gels, 
visualized by ethidium bromide staining and purified using Wizard PCR Preps DNA 
Purification System. The DNA concentration in each sample was determined using a 
Spectronic 1001 spectrophotometer (Milton Roy/Bausch & Lomb, Rochester, NY) and 
the purified PCR products were sequenced either directly or following TA cloning and 
plasmid purification. 



TA Cloning of PCR Products and Plasmid Purification 

PCR products were cloned in the pCR 2.1 vector using the TA Cloning kit 
(Invitrogen, San Diego, CA) according to the manufacturer's instructions. Briefly, PCR 
products were ligated in pCR 2.1 vector overnight at 14°C and INVaF' competent E. coli 
cells were transformed with the constructs. Cells were grown overnight at 37°C on LB 
plates containing 50 u-g/mL kanamycin and 40 mg/mL X-Gal, then liquid LB/kanamycin 
cultures were inoculated with positive colonies and incubated overnight with shaking 
(RPM=225) at 37°C, and plasmids were isolated using the Wizard Plus Minipreps DNA 
Purification System (Promega). Purified plasmids were visualized on ethidium bromide- 
stained agarose gels, the DNA concentration in each sample was determined using a 
Spectronic 1001 spectrophotometer and -0.3 ug were used for sequencing. 



24 
Sin gle-Strand Conformation Polymorphism (SSCP^ Analysis 

Detection of nucleotide changes by SSCP was performed as described by 
Aberanthy et al, 1997. Briefly, each PCR product was mixed with an equal volume of 
denaturing buffer and heated to 95°C for 3 min. The samples were loaded onto 0.8 mm 
thick, 1 0% native polyacry lamide gels which were run at ambient temperature at 9 W for 
6-10 h, depending on the size of the PCR product. Bands were visualized by silver 
staining (Beidler et al, 1982). 



DNA Sequencing and Sequence Analysis 

An ABI PRISM 310 Genetic Analyzer and dye terminator cycle sequencing kit 
(Perkin Elmer, Foster City, CA) with AmpliTaq DNA polymerase, FS were used for 
sequencing according to the manufacturer's instructions. Both strands of purified PCR 
products and plasmids were sequenced to confirm the accuracy of the results. Sequences 
were analyzed with the GCG Package (Devereux et al, 1984) and searches of the 
National Center for Biotechnology Information database were performed using the 
BLAST network server (Altschul et al, 1990) (National Library of Medicine, via 
INTERNET) and the Whitehead Institute Sequence Analysis Program (MIT, Cambridge, 
MA). For analysis of sequences, identified by the yeast two-hybrid method as potential 
interacting proteins, see the "Yeast Two-Hybrid Screens" section later in this chapter. 



25 
Allele-Specific Oligonucleotide Analysis 

PCR products spanning the mutation site in patient 371 were transferred to nylon 
membranes using a slot-blot apparatus. Approximately 5 ng of each PCR product was 
treated with a denaturing solution (0.5 M NaOH, 1.5 M NaCl), split in half and loaded in 
duplicate. Two 17mer oligonucleotides that span the region containing the mutation were 
synthesized. One contained the sequence of the normal allele (5'-CGCACATG 
GCAACCCTT-3'), while the other contained the sequence of the mutant allele (5'- 
GCACATGGGCAACCCTT-3'). These were end-labeled with [y- 32 P]dATP using T4 
polynucleotide kinase and hybridized to the membranes at 50°C. Hybridization and wash 
buffers were as described (Church and Gilbert, 1984). Membranes were washed 
sequentially at 45 °C, 55 °C and 65°C for 10 min each and exposed to X-ray film. 



PCR Amplification of Mouse Lyst cDNA Isoforms 

Total cellular RNA was isolated from mouse bone marrow cells by extraction 
with phenol/guanidine isothiocyanate (TRIzol Reagent). Five ug of RNA were reverse 
transcribed using Superscript II RNase H" reverse transcriptase, with first strand cDNA 
synthesis priming by an oligo(dT) primer and a gene-specific primer (5'-CTCGCAGAG 
CGGTGCTTATGTCCTGTG-3', 5'-CACAGTCATGGGACTGCTAA-3', for Lyst-l and 
Lyst-ll, respectively). One uL of cDNA was used as template for PCR amplification 
using KlenTaq DNA polymerase. PCR conditions were: 94°C for 1 min, then 30 cycles of 
94°C for 15 s, 68 °C for 10 or 5 min (for Lyst-l and Lyst-ll, respectively), followed by 20 



26 

or 10 min (for Lyst-l and Lyst-U, respectively) at 68 °C. The same upstream primer (5'- 
AGCGGAGGTGAAGCCTTATGCTGAGACAGT-3') was used for PCR amplification 
of both isoforms. Isoform-specific downstream primers used were 5'- 
TTATGTCCTGTGGGGACACTCCTTC-3' (for Lyst-l) and 5'-ACAGAGCATCC 
CCACTTCCCTATCTAAAGT-3' (for Lyst-ll). The 2 shorter mouse isoforms (Lyst-lll 
and Lyst-YV) were PCR amplified from mouse melanocyte cDNA using KlenTaq DNA 
polymerase. PCR conditions were: 94°C for 2 min, then 36 cycles of 94°C for 20 s, 56°C 
for 15 s, 68°C for 2.5 min, followed by 10 min at 68°C. PCR products were separated by 
electrophoresis on agarose gels and visualized by ethidium bromide staining. One uL of 
1 :500 dilution of PCR products was used as template for a nested PCR amplification with 
KlenTaq DNA polymerase. PCR conditions were: 94°C for 2 min, then 33 cycles of 94°C 
for 20 s, 56°C for 6 s, 68°C for 3 min, followed by 6 min at 68°C. PCR products were 
separated by electrophoresis on 1.2% low melting point agarose gels, visualized by 
ethidium bromide staining, excised from the gel with a sterile blade and purified using 
Wizard PCR Preps DNA Purification System. Purified PCR products were cloned in pCR 
2.1 vector by the TA method, and sequenced. 



Cloning of Human LYSTcDNAs 

Normal human bone marrow poly(A) + RNA was obtained from Clontech 
Laboratories and 1 ug was reverse transcribed using Superscript II RNase H" reverse 
transcriptase, with a ZTSr-specific primer (5'-CTTGTTGGCTAGTGCATATTGA 
CACAATCTTCC-3') used for first strand cDNA synthesis. The resulting ZTSr-specific 



27 

cDNA was used as a template for PCR amplification of the LYST coding domain with 
KlenTaq DNA polymerase in 50 uL reactions. PCR conditions were: 94°C for 2 min, then 
33 cycles of 94 °C for 20 s, 63 °C for 15 s (for some reactions) and 68°C for 10 min, 
followed by 12 min at 68°C. PCR products were separated on ethidium bromide-stained 
low melting point 1% agarose gels, excised with a sterile blade, purified using Wizard 
PCR Preps Purification System, and cloned in pCR 2.1 vector by the TA method. Some 
PCR products were treated with 10 U exonuclease I (United States Biochemicals) for 15 
min at 37 °C and reamplified by a nested PCR with KlenTaq DNA polymerase prior to 
cloning in the pCR 2.1 vector. The identity of the cloned products was confirmed by PCR 
amplification from purified plasmids with Z,F5T-specific primers using Taq DNA 
polymerase in 20-uL reactions. PCR conditions were: 94°C for 2 min, then 30 cycles of 
94°C for 20 s, 56 °C for 10 s, 72°C for 1 min, followed by 5 min at 72 °C. PCR products 
were visualized on ethidium bromide stained agarose gels and photographed. 

Segments of the human LYST cDNA sequence were obtained by an anchored, 
nested PCR (5'RACE-PCR) using liver cDNA (Clontech Laboratories) as a template, by 
RT-PCR using total RNA and by sequencing of human expressed sequence tags (EST) 
similar in sequence to mouse Lyst. For the 5'RACE-PCR, 2 nested primers were derived 
from a human EST (GenBank accession No. W26957) and had the following nucleotide 
sequences: 5'-CCAAGATGAAAGCAGCCGATGGGGAAAACT-3' and 5'- 
TCAGCCTCTTTCTT GCTCCGTGAAACTGCT-3'. For RT-PCR experiments, total 
RNA was prepared from the promyelocytic HL-60 cell line. RT-PCR was performed with 
Expand polymerase (Boehringer Mannheim) with the following primer pairs: 5'- 



28 

AGTTTATGAGTCCAAATGAT-3' and 5'-GAATGATGAAGTTGCTCTGA-3' (bp 
490-2034); 5'-CAGCAGTTCTTCAGATGGA-3' and 5'-ATCTTTCTGTTGTTCCC 
CTA-3' (bp 1891-3050); and 5'-TAGGGGAGCAACAGAAAGAT-3' and 5'-GCTCAT 
AGTAGTATCACTTT-3' (bp 3320-4722). The primers used to amplify the cDNA 
between base pairs 1891 and 3050 were derived from the mouse Lyst sequence. Human 
primers were designed from the sequence of the PCR product (1159 bp) and used to 
amplify the flanking sequences. Human LYST intron a' was PCR amplified from human 
genomic DNA (100 ng) with the primers 5'-CCGCTCGAGTAGGATCTT 
TAAGGTGAATAAC-3' and 5'-GTGATACTACTATGAGCCCTTCACAGTATC-3'. 
PCR conditions were: 94°C for 1 min, then 32 cycles of 94 °C for 15 s, 63 °C for 20 s and 
72 °C for 30 s, followed by 10 min at 72 °C. 



Molecular Probes 

The LYST2 probe was PCR amplified from a human fetal brain partial length 
cDNA clone (#32273), obtained from Image Consortium, using internal primers. 
KlenTaq DNA polymerase was used for amplification and the PCR conditions were as 
follows: 94°C for 2 min, then 36 cycles of 94 °C for 30 s, 54 °C for 15 s, 68 °C for 5 min, 
followed by 5 min at 68 °C. PCR product was then purified with Wizard PCR Preps DNA 
Purification kit. Other probes are described in the text or in the legends of the 
corresponding figures. All probes were radio-labeled by the hexanucleotide technique 
(Prime-It II random primer kit, Stratagene) with [oc- 32 P]dCTP (Amersham) according to 
the manufacturer's instructions. 



29 
Isolation of LYST2 cDNA Clones 

LYST2 clones were isolated by screening cDNA libraries using vector-insert PCR 
and hybridization. A human fetal brain cDNA library (Life Technologies Inc.) was 
screened using hemi-nested PCR with one vector primer and one Z,KST2-specific primer. 
One uL of cDNA library (5x1 9 cfu/ml) was used as template for PCR amplification with 
KlenTaq DNA polymerase. PCR conditions were: 94°C for 5 min, then 33 cycles of 94°C 
for 30 s, 63 °C for 30 s, 68 °C for 3.5 min, followed by 10 min at 68 °C. PCR products 
were separated by electrophoresis on agarose gels and visualized by ethidium bromide 
staining. PCR products were excised from low melting agarose gels, purified (Wizard 
PCR Preps DNA Purification kit), and cloned using the TA method. Resulting plasmids 
were purified with Wizard Plus Minipreps DNA Purification kit and sequenced. 

Membranes containing clones from a human fetal brain cDNA library (Clontech 
Laboratories) were screened by hybridization with a radioactively labeled cDNA probe 
from the 3 '-end of the LYST2 coding domain. Positive clones were detected by 
phosphorimaging (Molecular Dynamics), and purified plasmids were isolated and 
sequenced. Then new probes were PCR amplified from the 5 '-end of the novel cDNA 
sequence and the library screening was repeated in order to isolate overlapping clones 
extending the sequence in the 5' direction. 

A ZAP Express EcoRl mouse embryo cDNA library was screened by 
hybridization with the LYST2 probe. Identified cDNA clones were purified using Wizard 
Plus Minipreps DNA Purification kit and sequenced. 



30 
Southern Blot Analysis and Autoradiography 

Genomic DNA was isolated from mouse organs using standard techniques 
(Sambrook et al, 1989) or obtained from The Jackson Laboratory (Bar Harbor, ME). 
DNA was digested with restriction endonucleases from Promega (Madison, WI) or 
Boehringer Mannheim (Indianapolis, IN), and 10-ug samples were subjected to 
electrophoresis in 0.85% agarose gels for ~ 20 h at 35V. DNA was transferred to 
ZetaProbe (Bio-Rad Laboratories, Hercules, CA) or GenescreenPlus (DuPont Co., 
Wilmington, DE) membranes, which were neutralized in 2x SSC, UV-crosslinked for 1 
min and prehybridized at 65°C overnight. Hybridization to probes labeled by the 
hexanucleotide technique (Prime-It II random primer kit, Stratagene, La Jolla, CA) with 
[a- 32 P]dCTP (Amersham, Arlington Heights, IL) was performed at 65°C overnight. 
Membranes were washed in a solution containing 2x SSC, 0.1% SDS and 1 raM EDTA 
for 30 min at 65°C and then in a solution containing 0.5x SSC, 0.1% SDS and 1 mM 
EDTA for 5-15 min at 65°C. Blots were exposed to X-ray film (DuPont) in FisherBiotech 
(Pittsburgh, PA) autoradiography cassettes for 1-7 days and films were developed using a 
Konica QX60A processor. 



Northern Blot Analysis 

Isolation of poly(A) + RNA from fibroblast and Epstein-Barr virus-transformed B 
lymphoblastoid cell lines, formaldehyde agarose gel electrophoresis and northern blotting 
to ZetaProbe membranes were performed according to standard procedures (Sambrook et 



31 

al, 1989). Northern blots of 2 \ig poly(A) + RNA from various mouse tissues, human 
cancer cell lines and human lymphoid tissues, as well as a dot-blot containing poly(A) + 
RNA from 50 human adult and fetal tissues were obtained from Clontech Laboratories. 
The quantity of poly(A) + RNA on each dot was normalized for 8 housekeeping genes, 
and varied from 100 to 500 ng. Membranes were hybridized with various probes labeled 
with [a- 32 P]dCTP by the hexanucleotide technique (Prime-It II random primer kit). 
Hybridization conditions were the same as those employed with Southern blots. 
Autoradiography or phosphorimaging was performed using X-ray film or 
phosphorimager (Molecular Dynamics, Sunnyvale, CA), respectively. 



Simple Sequence Length Polymorphism (SSLP) PCR Amplification 

PCR reactions with Taq DNA polymerase (Boehringer Mannheim) were 
performed using 40 ng of mouse genomic DNA, 1 umol/L of each primer (Research 
Genetics, Inc., Huntsville, AL) and 200 umol/L of each dNTP in a 20 uL reaction. PCR 
conditions were: 95°C for 2 min, then 30-36 cycles of 94°C for 20 s, 55 °C for 30 s, 72 °C 
for 20 s, followed by 72 °C for 1 min. Amplification products were separated on 3% 
agarose gels, visualized by ethidium bromide staining and photographed. 



Backcross Mouse Panel and Genetic Mapping 

C57BL/6.LV X (C57BL/6.LV x CAST/EiJ)F, backcross mice were bred and 
maintained as described in Holcombe et al, 1991. Genomic DNA was isolated from 



32 

mouse organs using standard techniques (Sambrook et al, 1989) and Southern blots were 
prepared. The chromosomal assignment of each probe was determined by linkage 
analysis in 93 intersubspecific backcross mice. The 192 genetic markers (156 
microsatellite markers, 14 genes, 20 expressed sequence tags (Tchernev et al, 1997) and 
2 coat color loci) that have been previously mapped in this panel served as anchor loci 
with average genetic distance between them ~ 10 cM. Gene order was determined by 
minimization of crossover events and elimination of double crossovers between linked 
genes. Intergene distances were expressed in centimorgans (cM) and standard errors were 
determined as previously described (Green, 1981; Bishop, 1985). 



Cloning of LYST and LYST2 Bait cDNA Fragments in Yeast Two-Hybrid Vectors 

Fourteen overlapping cDNA fragments (with average size of ~850 bp each), 
covering the entire coding domain of LYST, were PCR amplified from LYST cDNAs 
cloned in the pCR 2.1 vector. LYST2 cDNA fragments were amplified from EST clone 
#32273, obtained from Image Consortium. Primer design ensured that all cDNA 
fragments would be amplified in frame and that known domains with potential functional 
significance would be preserved. All primers contained specific restriction sites at their 
5'-ends and all reverse primers contained stop codons near their 5'-ends. The sequence of 
all forward and reverse primers with their restriction sites, and the length and the position 
(according to GenBank accession number U67615 for LYST) of the amplified cDNA 
fragments are shown in Table 2 (for LYST) and in Table 3 (for LYST2). 



33 



a 
o 



o 

« 

BO 

e 

i 

CX) 

a 

< 

Q 



X) 



-a 
u 

55 

3 

09 

"3 

C 
'J 
i 



u 

3 

o 

'55 
3 

o 



•— 
DO 



D. 

U 

IX 



a 



a 

W 

N 



2| 

IX, C/2 



a. 

c 



K O 



oo 



in 

o 

© 






o 



in 

o 



ri 



o 
e'- 
en 
c\ 

oc 

<» 



o 
oc 



m 



ts 

oc 



cn 

o 

-t- 

i 

o 



00 



ri 

oo 

o 

o 



00 
00 



o 
o 
r- 

oo 



>n 
m 



>n 



m 

oo 



i 

O0 

>n 



oo 



oo 
m 
CN 
oo 
i 

r^ 
«<t 

r- 



ri 

oo 



m 

p 



oo 



oc 
m 



in 

oc 
m 
C* 

O 

m 

o 



oo 
oc 
O 



o 

in 

o 

i 

CN 

O 
in 

ON 



1/1 

m 

o 



o 



o 

>n 

o 



N 

g 



x 2 

S o 

£3 



I si 



V 2 



H 2 

5 © 
3 3 



S 2 

5 o 
£3 



53 2 

5 © 

c§£ 



1? 
£3 



cq "> 



1—1 h— . 
S © 

Cq C*3 



53 2 

S © 

c^3 



€ 2 
I 8 

Cq ^ 



c3 ^ 



= 5 
I 8 

Cq ^ 



1 2 

S © 
c§£ 



u 

a 

3 

cr 
u 

03 

T -2 

in 7 

« u 

W £ 

y o 

D . 

o a 

w g 

p* S" 
pq as 



y 

o 

u 

< 
a 

S a 
p < 



m 



o 

c 

3 



< 

r^ < 

O y 

u o 

uo 

< o 

U t_ 

o o 

^ < 



< o 

H < 



T3 

J3 



U 
O 

H 
H 
< 
U 
- 
< 
< 

a 

u 

- 
< 
< 
< 

u 

u 



a 
< 
u 

H 
O 

h u 



o a 

u 

< h 



g 

2 o 



o a 



W u 



< < 



2 a 



< 
< 
< 

< 



< 

< 
H 

H 
H 
< 
< 
< 

y 

a 
< 



H 
O 
r- 

< 
< 



a o 
o o 
y o 
a o 



u 
o 
o 

E- 
< 

a < 

fi 

§1 



o 
o 

t- 
u 



< h 
o < 
o < 
y o 
o o 



u b 

U y 

y h 

O y 
h h 

a h 

H 
O 

< o 
o a 
y y 
o o 



a 
< 
y 

H 
H 

o o 

y y 
y a 
y 

u 

a y 

P fc 

a < 
o < 
y a 
a a 



a 

H 

H 

H 
< 

i 

y 

a 



< 

< 

H 



a 
< t 
pb 



y 
y 


H 

a 
< 

r- 



y a 



<! h 

o < 
y w 



a o 

a < 

H O 

y a 
y < 

O y 

y H 

H y 






H u 



a o 
a a 
y y 

a o 



y 

E- 

y 

< 
y 
y 
y 

H 
< , 

2 < 
o S 



y y 





< y 

< a 

< a 

< < 

o o 

o y 

^ < 
P < 
b < 
% < 

O o 

< a 

o u 

O H 

P 2 

y o 

y < 

y y 

y h 



a 

y 2 
y o 

< H 






y a 

H O 

E < 

9 2 



y 

< 

y 
a 

a 



Ou 



< 2 
a a 

< y 
y o 

< o 

y < 
P y 



2 o 
o a 

y y 
o a 



y y 
y p 



U y 

y h 



a a 
a o 
y y 
a a 



a < 
P < 
2 o 
o a 
y y 
o o 



2 2 

a a 

y y 

a a 



y a 
< a 
y 
y 

< 

E- 
< 

a 

E- 
< 
O 
E- 
< 

a 

E- 

o 
y 

H 
H 

a 
a 
< 
< 

y 
y 
y 

E- 

< 

a 
a 

H 

2 o 

o a 
y y 

a a 



2 
a 
a y 

< < 



o o 

y < 
y y 
y h 



E- 
< 

2 

a 

H 

a a 
a o 
y y 
a a 



H 

a 



a 






*N 






E-. 



E~, 



^3^0 






^3 oo 



^^ 









>-i ' — i 



--1 






34 





S 


a 












2 


N 


0\ 


en 

00 


o 
in 






fa 


5o 












H 
Z 


« 

a. 






^ 




U 


g 


z 


Tj" 


^O 


(N 


^ 




S b 
op 




00 


t 


i 






O 

PL* 


m 


CO 




CO 


6 


























E 

o 

en 

d 




fa 

>- 

N 
Z 


v 2 


v 2 


« 2 




OJ 


w 










e 














od 














1 














< 














2 














Q 














o 















-4-J 




cu 

u 

c 
o 














3 

cr 


U 

^ fa 


U 


<2 


o 


rt 


m 


£ 


3 a 

is 




< o 


o 

1-' 


t 


_o 


^ < 


T3 




1 


o 5 




o u 




LT] 


1 


9 8 


V. < 


3 

GO 


u 




3g 


< u 




T3 

C 

1 


z 


> 


< u 


< H 


g 
p 


>- 






o < 


lin 


a 


o 


fa fa 


H fa 


H < 


H 


w 


c 


< b 


< < 




< h 


'5 


m 


3 


o < 


O U 


o b 


-3 


& 


a- 


h a 


H U 


< a 


h ^2 


s 


m 


Vj 


u u 


O fa 


u a 


u o 


o 


5 


i- 


h o 


h < 


< a 


H O 


03 

-4— » 


U 

c 

Q. 


y^ 




2 < 


^ 3 


'<*> 


cu 


3 


|3 


^ fa 


a u 


^ o 


5 




1 


2 < 


2 < 


P < 


_o 




-a 


fe u 


fc u 


fc o 


fc u 


*4-> 




« 


"5 H 


9 H 


5 H 


^5 H 


'C 






< u 


< U 


< U 


< u 


<-> 

c/5 




£ 


fa fa 


H H 


H H 


H H 


<L> 




U H 


U H 


O H 


U fa 


1-H 




^-^ 


< < 


< < 


< < 


^^ 






< < 


< < 


< < 


^ 






a o 


o o 


o a 


o o 








o 


o o 


a a 


o o 


E 






u u 


u u 


u u 


u u 


E 






a a 


o a 


a a 


o a 


O. 




% 










(* 




fa 










u 

fa 


ttf 

z 


Z 

w 


p 


R 


p 


p 




2 
a 

2 


So -i 


SO <N 


So "o 


So > * 


ro 


fa 


5s 


>>, 


K 


K 


4) 


a 


M 


M 


M 


<i 


H 




fa 











p 



o 
a 






> 

| 

u 
a 

(U 

CO 

3 

O 
• — h 
+-* 

'co 

o 

3 

aj 

E 
S3 



o 
o 

3 



U 

o 

3 
U 

3 

cr 
u 

< 
z 

Q 

o 

p 

u 

J-. 

'-3 
3 
U 
U 

-3 

aj 
O 

3 



u 
o 

3 

s 

cr 
u 



35 

KlenTaq DNA polymerase and 12.5 pmol of each primer were used in each 50 uL 
amplification reaction. PCR conditions were: 94°C for 5 min, then 30 cycles of 94 °C for 
40 s, 50 °C for 40 s, 72 °C for 2 min, followed by 72 °C for 5 min. PCR products were 
visualized on ethidium bromide-stained agarose gels, purified using QIAquick PCR 
purification kit (QIAGEN Inc., Valencia, CA) and digested with the corresponding 
restriction enzymes (Tables 2 and 3) in 20 uL-reactions overnight at 37 °C. Digested PCR 
products were excised from 1 % low melting point agarose gels, purified using QIAquick 
gel extraction kit (QIAGEN Inc.) and ligated in the yeast Gal4 activation domain cloning 
vector pAD-GAL4 (modified pGAD GH, Clontech Laboratories) and in the yeast Gal4 
DNA-binding domain vector pGB-GAL4 (modified pGBT9, Clontech Laboratories). 
DH10B competent cells were transformed with the purified constructs by electroporation, 
incubated for 1 h at 37 °C with shaking (RPM=150) and plated on LB/ampicillin (50 
jag/mL) plates containing X-Gal and IPTG. The identity of cloned fragments was 
confirmed by PCR of bacterial colonies with one vector primer and one fragment-specific 
primer under the same amplification conditions as above. LB/ampicillin liquid cultures 
were inoculated with positive colonies and incubated overnight at 37 °C with shaking. 
Purified plasmids were isolated using QIAprep Spin Plasmid kit (QIAGEN Inc.) and 
visualized on ethidium bromide-stained agarose gels. All inserts were sequenced to 
ensure that PCR amplification reproduced an accurate copy of the LYST and LYST2 
sequence and that the cDNA fragments were cloned in frame. 



36 

Yeast Two-Hybrid Screens 

Two types of screening for protein-protein interactions were performed by the 
yeast two-hybrid method. In a "forward" screen, one hybrid consisted of the DNA 
binding domain of the yeast transcriptional activator Gal4 fused to a "bait" portion of 
LYST or LYST2. The other hybrid consisted of the Gal4 activation domain fused to 
"prey" sequences encoded by a mammalian cDNA library. In a "reverse" screen, the 
"bait" part of LYST or LYST2 was fused to the Gal4 activation domain, and the "prey" 
sequences of the mammalian cDNA library were fused to the Gal4 DNA binding domain. 
The prey cDNAs were obtained from a human fetal brain cDNA library of 1 x 10 7 
independent isolates (Clontech Laboratories). The library was synthesized from 
JL77oI-(dT) 15 -primed fetal brain mRNA (pooled from five male/female 19-22 week fetuses) 
that was directionally cloned into either pAD-GAL4, an yeast Gal4 activation domain 
cloning vector including the LEU2 gene for selection in yeast deficient in leucine 
biosynthesis, or pBD-GAL4, an yeast Gal4 DNA-binding domain cloning vector 
including the TRP1 gene for selection in yeast deficient in tryptophane biosynthesis. 

To ensure that the bait DNA-binding domain fusions do not possess intrinsic 
transcriptional activity, a test for "bait self-activation" was performed. YULH yeast cells, 
transformed by the lithium acetate/polyethylene glycol method (Ito et al, 1983) with the 
pBD-GAL4 bait constructs, were grown at 30°C on SC-Trp medium to select for the 
presence of the DNA-binding domain plasmid. The selected yeast cells were then plated 
on SC-Ura medium, grown overnight at 30°C, and examined for growth. Cells that did 
not grow contained baits which were not "self-activating" proteins, that is, these proteins 



37 

required interaction with a second protein domain in order to form a functional complex 
that can activate the transcription of reporter genes. Baits from non-growing cells were 
used for further screening. 

In the forward screens, the constructs encoding the baits were introduced by 
lithium acetate/polyethylene glycol transformation into the yeast strain YULH (mating 
type a, ura3, his3, lys2, Ade2, trpl, leu2, gal4, gal80, GAL1-URA3, GALl-lacZ), while 
the prey sequences were transformed into the yeast strain N106r (mating type a, ura3, 
his3, ade2, trpl, leu2, gal4, gal80, cyh', Lys2::GALl UAS -HIS3 1ATA -HIS3, 
ura3::GALl UAS -GAL TATA -lacZ). For the reverse screens, baits were transformed into N106r 
and preys into YULH. Initially, a limited screening using approximately 50,000 prey 
library members was performed to ensure that bait fusions do not interact promiscuously 
with many proteins (test for non-specific binding). Only baits that did not possess 
intrinsic transcriptional activity (i.e., passed the test for "bait self-activation") and that did 
not interact indiscriminately with numerous proteins (/. e. , passed the test for non-specific 
binding), were used for screening the entire prey library. In these full-scale screens, the 
two transformed populations were mated using standard methods (Sherman et al, 1991). 
Briefly, cells were grown at 30°C until mid-to-late log phase on media that selected for 
the presence of the appropriate plasmids, i.e. yeast containing activation domain 
constructs were grown on SC-Leu medium, while cells containing DNA binding domain 
plasmids were grown on SC-Trp medium. The two mating strains, a and a, were then 
diluted in YPAD media (Sherman et al, 1991), filtered onto nitrocellulose membranes, 
and incubated at 30°C for 6-8 hours. The cells were then transferred to media selective for 



38 

the desired diploids, i.e., for yeast harboring reporter genes for beta-galactosidase, uracil 
auxotrophy, and histidine auxotrophy, and for expression of the vectors encoding the bait 
and prey. The mating products were plated on SC (synthetic complete) media (Kaiser et 
al, 1994) lacking adenine and lysine (to select for successful mating), leucine and 
tryptophane (to select for expression of genes encoded by the bait and prey plasmids), 
and uracil and histidine (to select for protein interactions). This medium is referred to as 
SCS medium, for SC Selective medium. 

Selected clones were tested for expression of P-galactosidase to confirm the 
formation of an LYST:LYST-interacting protein (IP), resp. LYST2:LYST2-IP, 
interactions. Filter-lift P-galactosidase assays were performed as modified from the 
protocol of Breeden and Nasmyth, 1985. Colonies were patched onto SCS plates, grown 
overnight, and replicated onto Whatman No.l filters. The filters were then assayed for 
P-galactosidase activity. Colonies that were positive turned a visible blue, indicating that 
a protein-protein interaction has occurred, enabling the transcription of the reporter gene 
LacZ. 

Cells in colonies positive for protein interaction contained both DNA-binding and 
activation-domain plasmids. These cells were regrown as single isolates in individual 
wells of 96-well plates. Ten microliters of each isolate were lysed, the inserts within the 
pAD-GAL4 and pBD-GAL4 plasmids were amplified by PCR using primers specific for 
the insert- flanking sequences of the vectors, and approximately 300 amino-terminal bases 
of each insert were determined using an ABI 377 sequencer. Comparison to known 
sequences was made using the BLAST program publicly available through the National 



39 

Center for Biotechnology Information (N. C.B.I.)- The assembly and identity searches of 
the sequences encoding human EST were performed by using publicly available EST 
assembly databases such as the N. C.B.I. BlastN 2.0 program (Altschul et al, 1990). 
Sequences that aligned with 95% or greater identity at the nucleic acid level over their 
termini of at least 30 nucleotides were utilized if the alignment resulted in 5' extension or 
3' extension of the EST sequence. Once this first assembly was complete, the extended 
sequence was again subject to the BlastN comparison to detect new homologies to the 
added extensions. The sequence was extended in both directions until new related 
sequences that allowed extension of the assembled sequence were no longer detected. The 
assembled EST sequence was subjected to further searches using the BlastX 2.0 program 
for the identification of protein coding regions by database similarity search (Gish and 
States, 1993). The BlastX software translates the DNA sequence in all six reading frames 
and compares the translated protein sequence with those in protein databases. The 
statistical significance is estimated under the assumption that the equivalent of one entire 
reading frame in the query sequence codes for protein and that significant alignments will 
involve only coding reading frames. Furthermore, the sequences were analyzed for open 
reading frames using software that translates the assembled DNA sequence in all six 
reading frames using the standard genetic code. The interacting ESTs were obtained from 
directionally-cloned libraries, and thus the direction of translation of the assembled EST 
is known as 5' to 3' and the open reading frame is also known. ORFs longer than 50 
amino acids following an initiator codon or an ORF with no initiator methionine encoded 
at the 5' -end were determined to be possible protein products, and were compared to 



40 

sequences in protein data bases using the BLASTP 2.0 program (Altschul et al, 1990). 
Further protein sequence analysis was performed after selecting a suitable open reading 
frame. The protein sequence was compared to previously characterized protein domains 
present in the BLOCKS and PRODOM motif databases (Bairoch, 1992; Henikoff and 
Henikoff, 1991; Nakai and Kanehisa, 1992; Wallace and Henikoff, 1992). 



Confirmation of the Specificity of Protein Interactions by the Yeast Two-Hybrid System 

In addition to sequencing all baits, and the tests for "bait self-activation" and for 
non-specific binding, described in the previous section, several other tests were 
performed to determine the specificity of the detected bait:prey interactions. In the test for 
"prey self-activation", YULH yeast cells were transformed with individual plasmids 
encoding the DNA-binding domain fusions of all preys that were detected to interact with 
LYST or LYST2. The transformed haploid yeast cells were grown at 30°C on SC-Trp 
medium to select for the presence of the DNA-binding domain plasmid. The selected 
yeast cells were plated on SC-Ura medium, grown overnight at 30°C, and examined for 
growth. Cells that did not grow contained preys which were not "self-activating" proteins, 
that is, these proteins required interaction with a second protein domain in order to form a 
functional complex that can activate the transcription of reporter genes. Preys from non- 
growing cells were used in the other confirmation tests. 

YULH and N106r haploid yeast cells, transformed with DNA-binding domain, 
resp. activation domain fusions of preys that were detected to interact with LYST or 
LYST2, were grown on SC-Trp or SC-Leu media to select for the corresponding 



41 

plasmids. Filter-lift P-galactosidase assay was performed on the selected haploid cells. 
Colonies that did not turn blue contained preys that needed to interact with another 
protein in order to activate the transcription of the LacZ reporter gene. Only cells that did 
not grow on SC-Ura medium {i.e., passed the test for "prey self-activation") and that did 
not turn blue {i.e., passed the P-galactosidase test for haploids) were used in the next 
confirmation tests. 

The following "matrix mating" test was performed to duplicate the initial forward 
and reverse screens and to determine whether the same protein-protein interactions would 
be detected. YULH yeast cells, transformed with DNA-binding domain fusions of baits 
(forward screen) or preys (reverse screen), and N106r cells, transformed with activation 
domain fusions of baits (reverse screen) or preys (forward screen), were grown in YPAD 
medium at 30°C. The "matrix mating" was performed by spreading yeast containing 
individual baits on YPAD plates, transferring (in 96-well format) prey-containing cells 
onto the spread baits, and incubating the plates overnight at 30°C. Each plate contained 
also positive (proteins that are known to interact) and negative (proteins that do not 
interact with a particular bait) controls. Resulting diploid colonies were replica-plated on 
2 types of media: on SCS, which selected for successful mating, for protein-protein 
interactions and for presence of plasmids, and on SC-Leu-Trp plates, which were used for 
a filter-lift p-galactosidase assay. Only baits and preys from diploid yeast cells that were 
able to grow on SCS medium {i.e., passed the "matrix mating" test) and that turned blue 
{i.e., passed the P-galactosidase test for diploids), were PCR amplified with vector 
primers and the resulting PCR products were sequenced. If sequencing determined that 



42 

the identity of a particular bait: prey pair matched that obtained after the initial screens, 
the interaction was considered confirmed by the yeast two-hybrid method. An overview 
of the described yeast two-hybrid screening and confirmation procedures is shown in 
Figure 2. 



CLONE LYST AND LYST2 BAITS IN 

ACTIVATION- AND BINDING-DOMAIN VECTORS 

I 

SEQUENCE ALL CONSTRUCTS 

I 

TRANSFORM IN YEAST 

I I 

TEST FOR TEST FOR 

BAIT SELF-ACTIVATION NON-SPECIFIC BINDING 

i I 

FORWARD AND REVERSE SCREENS 

I 

P-GAL. TEST FOR DIPLOIDS 

i 

PCR INSERTS 

I 

SEQUENCE INSERTS 

I 

DETERMINE IDENTITY OF BAIT:PREY INTERACTIONS 

I 

RE-TRANSFORM YEAST 

I I 

TEST FOR PREY SELF-ACTIVATION P-GAL. TEST FOR HAPLOIDS 

I I 

MATRIX MATING 

i 

P-GAL. TEST FOR DIPLOIDS 

I 

PCR INSERTS 

i 

SEQUENCE INSERTS 

I 

DETERMINE IDENTITY OF BAIT:PREY INTERACTIONS 

I 

COMPARE TO INITIAL IDENTITIES 

I 

INTERACTION CONFIRMED BY THE YEAST TWO-HYBRID METHOD 

Figure 2. Overview of the screening and confirmation procedures by the yeast 
two-hybrid method. 



RESULTS 



Identification of Mutations in Patients with Chediak-Higashi Syndrome and in Beig e 

Mice 

Detection of mutations in the CHS gene of human patients and beige mice was 
undertaken for three main reasons: 

To provide additional evidence that LYST, resp. Lyst is indeed the gene 
responsible for the Chediak-Higashi syndrome in human and for the beige 
mutation in mouse 
- To identify critical amino acids and domains that are essential to the function 
of the CHS gene 

To determine whether a genotype-phenotype correlation could be established 
between different genetic defects and specific disease manifestations. 
A summary of mutations detected in patients with Chediak-Higashi syndrome is 
shown in Table 4. Several different approaches were used for mutation detection. As an 
initial screen for mutations, we analyzed northern blots of poly(A) + RNA from CHS 
patients. The largest mRNA species (-13 kb) was greatly reduced in abundance or absent 
in lymphoblastoid mRNA of patients PI and P3, respectively (Fig. 3 A), while the smaller 
transcript (~4.4 kb) was present and undiminished in abundance in all three patients. 



43 



44 



B 



12kb- 



4.4kb-l 




I Q CA AC C C T T.< 

670 680 




CCT TQTCCAlQOT TQAQQA 
690 




373 



Figure 3. Mutation analysis in CHS patients. (A) Northern blot of 2 ug aliquots of 
lymphoblastoid poly(A) + RNA from CHS patients and a control. The probe used for 
hybridization corresponds to nucleotides 490-8 1 7 of the CHS cDNA (GenBank accession 
No. U70064). Exposure time for autoradiography was 48 h. (B) SSCP analysis of cDNA 
corresponding to nucleotides 439-806 of the CHS cDNA. Each lane contains samples 
from individual patients as indicated. Extra bands in lanes corresponding to patients 371 
and 373 are denoted with an arrow. (C) Sequence chromatograms showing mutations in 
cDNA clones from patients 371 and 373. The upper part is normal human CHS cDNA 
sequence. The arrows indicate the positions of a G insertion (coding domain nucleotide 
118, patient 371), and C->T substitution (coding domain nucleotide 148, patient 373). 
The sense strand is shown. 



45 

While the selective loss of the larger transcript in these two patients cannot be 
excluded definitively, rehybridization of this blot with an actin probe suggested that the 
absence of the larger transcript was not due to uneven gel loading or RNA degradation. 
Fibroblast poly(A) + RNA from three other CHS patients (369, 370 and 373) showed a 
moderate reduction in the ~13 kb mRNA (51-60% of control by densitometry), while the 
-4.4 kb mRNA was essentially unaltered in abundance (103-147% of control). 

Single-strand conformation polymorphism (SSCP) analysis was undertaken using 
cDNA samples derived from lymphoblastoid or fibroblast cell lines from CHS patients, in 
collaboration with Drs. Steve Colman and Margaret Wallace. Anomalous bands were 
detected in PCR products from the 5 '-end of the open reading frame in two unrelated 
CHS patients (371 and 373) that were different from those with probable selective loss of 
the larger transcript on northern blots (Fig. 3B). Sequence analysis identified a C-»T 
substitution at nucleotide 148 of the coding domain in patient 373 (Fig.3C). Four of the 
nine cDNA clones derived from patient 373 contained this mutation. Restriction enzyme 
digestion confirmed the mutation: Taql digestion of cDNA (nucleotides 520-808) showed 
loss of this restriction site in patient 373 to be heterozygous. The C->T substitution 
creates a stop codon at amino acid 50 (R50X). The mutation in patient 373 occurs within 
a CpG dinucleotide, which, when the cytosine is methylated, represents a common 
hotspot for mammalian mutations due to deamination. 

In patient 371, a G insertion was found at nucleotide 118 of the coding domain, 
resulting in a frame shift at codon 40 and termination after amino acid 62 (Fig. 3C). 
While analysis of genomic DNA had shown patient 371 to be heterozygous for this 



46 

mutation (Nagle et al. , 1 996), each of five cDNA clones isolated from lymphoblasts of 
this patient contained the G insertion. Allele-specific oligonucleotide hybridization of 
cDNA from this patient failed to detect a signal with an oligonucleotide corresponding to 
the normal allele, suggesting that the patient is transcriptionally hemizygous for the G 
insertion. Alternatively, the lack of allele-specific oligonucleotide hybridization may 
reflect a base mismatch (such as a polymorphism) within the patient's cDNA. 

Mutations were identified in three other CHS patients by cloning and sequencing 
fragments of their LYST coding domains. A homozygous C-»T substitution at nucleotide 
3085 of the coding domain creating a stop codon at amino acid 1029 (Q1029X) was 
found in patient 370. In patient 369, a heterozygous frameshift mutation was detected. 
Nucleotides 3073 and 3074 of the coding domain were deleted in two of five cDNA 
clones, resulting in a frameshift at codon 1026 and termination at codon 1030. A 
homozygous C-»T substitution at nucleotide 3310 of the coding domain was identified in 
patient 372. This mutation created a stop codon at amino acid 1103, resulting in 
premature termination. Despite mutation analysis of >6 kb of open reading frame, the 
mutations in CHS patients PI and P3, that may have a selective loss of the larger 
transcript on northern blots, have not been identified. 

Lymphoblasts from all of these patients (369, 370, 371, 373, PI and P3) contain 
the giant perinuclear lysosomal vesicles that are the hallmark of the Chediak-Higashi 
syndrome. The parents of patients 369 and 370 are known not to have been 
consanguineous. Patients 369, 370 and 371 had typical clinical presentations of CHS, 
with recurrent childhood infections and oculocutaneous albinism. In contrast, the clinical 



47 



course of patient 373 was milder - this patient has not had systemic infections and 
remains alive at age 37. Patient 373 does, however, have hypopigmented hair and irides, 
as well as peripheral neuropathy. A summary of the described mutations, detected in 
patients with Chediak-Higashi syndrome, is shown in Table 4. 

Table 4. Mutations identified in patients with Chediak-Higashi syndrome. 



PATIENT 


ZYGOSITY 


MUTATION 


CONSEQUENCE 


369 


Heterozygous 


bp 3073 & 3074 deleted 


Codon 1 026 frameshift, 
stop at codon 1030 


370 


Homozygous 


C3085T 


Q1029X 


371 


Heterozygous 


G insertion at bp 118 


Codon 40 frameshift, 
stop at codon 62 


372 


Homozygous 


C3310T 


stop at codon 1 1 03 


373 


Heterozygous 


C148T 


R50X 


X 

Naglee/a/., 1996 


Homozygous 


bp 1467 deleted 


Codon 489 frameshift, 
stop at codon 566 


PI 


? 


Ul3.5kbmRNA 


lack of protein 


P3 


? 


Ul3.5kbmRNA 


lack of protein 



48 



Mutations were also identified in several beige alleles. A 5-kb genomic deletion 
that contained the 3 '-end of Lyst exon (3, and exons y and 5, was found in bg" J DNA. This 
mutation was detected by hybridization of Lyst-derived probes to Southern blots of 
genomic DNA from all potential progenitor mouse strains (Fig. 4), and by PCR 
amplification of several Lyst fragments (Fig. 5). This deletion corresponds to a loss of 
approximately 400 internal amino acids of the predicted Lyst protein. Furthermore, 
whereas the 5'-end of the bg" J deletion occurs within Lyst exon P, the 3'-end is intronic. 
Therefore the truncated Lyst mRNA in bg" J mice is also anticipated to splice incorrectly, 
terminate prematurely, and lack polyadenylation. Unfortunately, it was not possible to 
confirm this prediction since bg" J mice are extinct and only archived genomic DNA was 
available. 

Quantitative reverse transcription (RT)-PCR demonstrated a moderate reduction 
in Lyst mRNA in bg and bg J liver, and a gross reduction in bg 23 {Lyst AOD after 
normalization for p-actin mRNA: +/+, 1.00; bg 2J /bg 2J , 0.19; bg/bg, 0.28; bg J /bg J , 0.40) 
(Fig. 6). A commensurate reduction in bg 23 transcript abundance was noted by using 
several primer pairs derived from different regions of the Lyst cDNA. Aberrant Lyst RT- 
PCR products were not observed. The particularly striking (more than fivefold) reduction 
in Lyst expression evident in bg" homozygotes suggested the existence of a mutation in 
bg 2J that results in decreased transcription or mRNA instability. The molecular basis of 
the reduction in Lyst mRNA in bg 23 is not yet known. 



49 



A 



*> 



B 

4* & 



& 



4* 



QA- 



<A***$W^ 



■MMN 




Hind 111 Mspl Taql Hindm Mspl Taql Hind III Mspl Taql 



Figure 4. Southern blot identification of an intragenic Lyst deletion in bg" J . A 
Southern blot was sequentially hybridized with 3 Lyst probes: (A) the probe (nucleotides 
1262-3433 of Lyst cDNA) extends upstream of the bg" J deletion. (B) the probe 
(nucleotides 2835-3433 of Lyst cDNA) is completely deleted; (C) the probe (nucleotides 
3594-4237 of Lyst cDNA) extends downstream of the bg" J deletion. Restriction 
endonucleases are indicated at the bottom of each panel, and molecular size standards (in 
kb) are shown to the left. Southern blots were prepared from genomic DNA of all 
potential progenitor mouse strains, but only C57BL/10J, C57BL/6J and C57BL/6J- bg" J 
are shown. 



50 



0.6 - 



4*i4W 






B 



o ** 

♦ 



V £| 



*>* 



fAM^f 




#J> 



y > 



d — n 




* i 



, 1.9-kb 



D 



1.6-kb 



1 t v 



0.9-kb 



\~-bg 11 J deletion +i 



2.1 -kb 






S3 



Figure 5. PCR analysis of the bg" J deletion and genomic structure of Lyst in the 
vicinity of the deletion. C57BL/10J, C3HeB/FeJ, C57BL/6J and C57BL/6J- bg" J 
genomic DNA and Lyst cDNA were used templates in the PCR reactions. Amplicons 
illustrated correspond to: (A) Lyst cDNA nucleotides 1337-1837, which represent exon p 
and are upstream of the deletion; (B) nucleotides 2670-3210, which represent exon y, 
deleted in bg" J DNA; (C) nucleotides 4913-5433, which represent an exon downstream 
from the deletion. No amplicon was observed in control PCR reactions performed 
without template. More than 30 other STSs that had been localized within the bg non- 
recombinant interval amplified normally from bg" J DNA. (D) Genomic structure of Lyst 
in the vicinity of the bg" J deletion. Lyst exons (a, p\ y, 5, e and <(>) are depicted by black 
boxes, and intervening introns by a solid line. Nucleotides of the mouse Lyst cDNA that 
correspond to exonic boundaries are indicated above the boxes. The 3' end of exon p, and 
all of exons y and 8, are deleted in bg" J DNA. Genomic structure and intronic sequences 
were ascertained by sequence analysis of nested PCR products, performed with exonic 
primers and PI genomic clone as template. Boundaries of the bg" J deletion were 
determined by PCR of genomic DNA. 



51 



x \ x # $ $ ^ 



Lysf 



4cfo 



%of+/+ 100 40 
(normalized) 




19 28 



Figure 6. Quantitative reverse transcription (RT)-PCR analysis of Lyst in several 
bg alleles. Lyst mRNA from bg 1 , bg 2 - 1 , bg and control livers was reverse transcribed, PCR 
amplified and the amount of the resulting PCR products was quantitated by measuring 
AOD after normalization for p-actin mRNA (% AOD for Lyst are shown below the gel 
image). 



52 



Identification and Characterization of Lyst mRNA Isoforms 

The investigation of mRNA isoforms of the CHS gene was undertaken for the 
following reasons: 

- To resolve the discrepancy between the initial reports about the identification 

of the beige gene. The partial cDNA sequence, published by Perou et al. , 

1996b, did not match the complete open reading frame, reported by Barbosa et 

al, 1996. 

To identify the isoform of primary functional significance, as well as other 

alternatively spliced variants that may have some importance. 

To determine which of the predicted domains are crucial for the function of 

the CHS protein. 
The existence of more than one mRNA isoform of the mouse beige gene 
(designated Lyst) had been suggested by northern analysis (Fig. 7) and by the two 
different Lyst cDNA sequences that were reported. Northern blots indicated that Lyst is 
ubiquitously transcribed, both temporally and spatially, in mouse and human tissues. 
Complex alternative splicing, with both constitutive and anatomically restricted Lyst 
mRNA isoforms, was also revealed. The largest Lyst transcript in human and mouse was 
12-13 kb and additional smaller alternatively spliced variants were also present with 
varying abundance in different tissues (Fig. 7). 



53 



A B 



*• W <fWW ** V<* WJT&FK* 



9.5 
7.5 
4.4 

2.5 
1.4 




Mid. Lyst 









9.5 
7.5 

4.4 

2.5 

1.4 




5' LYST 



5' LYST 



Figure 7. Northern blot analysis of mouse and human ZF5T. (A), (B): northern 
blots of 2 ug poly(A) + mRNA from various mouse tissues hybridized with probes that 
correspond to (A), nucleotides 4423-4631, and (B), nucleotides 1430-2457 (exon P) of 
mouse Lyst cDNA. (C), (D): northern blots of 2 jig poly(A) + mRNA from (C), human 
lymphoid tissues, and (D), human cancer cell lines, hybridized with a probe that 
corresponds to nucleotides 357-800 of human IKSTcDNA. Molecular size standards (in 
kb) are shown to the left. 



54 

Mouse Lyst cDNA isoforms were identified by anchored nested PCR (3'-RACE- 
PCR). Two fragments (1.25 kb and 2 kb) were amplified from mouse spleen cDNA using 
this technique. The 1.25 kb clone contained the 3'-end of the previously described 5893 
bp Lyst cDNA that corresponds to a small mRNA isoform (Lyst-ll). The 5 '-end of the 2 
kb clone, however, contained sequence derived from the Lyst-ll cDNA, while the 3' -end 
sequence was from the largest isoform (Lyst-l, previously called BG) of the beige gene. 
Reverse transcription (RT)-PCR confirmed that nucleotides 1-4706 of Lyst are common 
to both mRNA isoforms (Fig. 8A). The large isoform cDNA that contains the entire 
coding domain was assembled from nucleotides 1-4706 of Lyst, the 2 kb 3'RACE-PCR 
clone, and 6824 nucleotides of BG cDNA. This 11 817 bp cDNA sequence {Lyst-l, 
GenBank accession number U70015) corresponds to the largest mRNA observed on 
northern blots (Fig. 7). This ~1 1.8 kb cDNA , however, is truncated at both the 5'-end of 
the 5 '-untranslated region (UTR) and at the 3 '-end of the 3'-UTR, and thus is smaller 
than the largest mRNA (-12-13 kb), observed on mouse northern blots. 

Analysis of a PI genomic clone (number 8592), containing the entire beige gene, 
revealed that the 1 1,817 bp Lyst-l cDNA results when splicing occurs from Lyst exon a 
(containing nucleotide 4706) to downstream exon x (Figs. 8 and 10). In contrast, 
incomplete splicing and reading through intron a' (interposed between exons a and x) 
yields the truncated Lyst-ll isoform, of length 5893 bp cDNA (Lyst-ll, Fig. 8, GenBank 
accession number L77884). Lyst intron a' encodes 37 in-frame amino acids followed by a 
stop codon and a polyadenylation signal. Lyst-ll corresponds to one of several smaller 
mRNAs observed on northern blots (Fig. 7). Full-length cDNAs corresponding to the 



55 

Lyst-l and Lyst-ll isoforms were both amplified from mouse bone marrow RNA by RT- 
PCR. The putative Lyst-I and Lyst-II proteins are of relative molecular mass 425 287 (M r 
425 kDa) and M,. 172.5 kDa, respectively. 



a 


j^r 




Lyst-l 11.8kb | 


■ 


m 


Exon-I 


Exon-II 




Intron-I' 




4706 




Lyst-ll 5.9kb 




■1 



Lyst-ll Lyst-l 

•4 *- ■* *■ 

RNA DNA RNA H,0 



1353 

1078 

872 




F1/R1 F1/R2 



Figure 8. Alternative splicing of the CHS gene. (A) Alternative splicing of mouse 
Lyst. Solid lines represent Lyst exons a and t (not drawn to scale). Splicing of exon a to 
exon x occurs in the Lyst-l mRNA (-12 kb). The hatched box represents the intronic 
region that forms the 3' end of the Lyst-ll ORF (5.9 kb). The intron contains a stop codon 
(*) and a polyadenylation signal (A). Nucleotide positions indicated are from GenBank 
accession No. L77884 (Lyst-ll) and U70015 (Lyst-l). (B) Detection of mRNA isoforms of 
mouse Lyst by RT-PCR and genomic PCR. DNase-treated mouse melanocyte RNA was 
reverse transcribed and amplified with primers Fl/Rl (expected amplicon size 273 bp) or 
F1/R2 (expected amplicon size 560 bp). RNase-treated C57BL/6J DNA was amplified 
with primers Fl/Rl. 



56 

cDNAs corresponding to the human homolog of the largest mRNA isoform of the 
beige gene were obtained by identification of human expressed sequence tags (ESTs) 
similar in sequence to mouse Lyst-\ by database searches (GenBank accession numbers 
L77889, W26957 and H51623). Intervening cDNA sequences were isolated using RT- 
PCR, and a partial human cDNA sequence (GenBank accession number U70064; 7. 1 kb) 
was assembled by alignment of these clones with the largest mouse bg gene cDNA. The 
predicted human and mouse peptides shared 82% identity over 1990 amino acids. The 
predicted human amino acid sequence contains a six amino acid insertion at residue 1039, 
relative to that of mouse. The complete sequence of the human CHS cDNA was reported 
by Nagle et al, 1996. This 13.5 kb cDNA sequence corresponds to the largest mRNA 
observed on northern blots from human tissues (CHS-l, Fig. 7 and inset of Fig. 9). These 
northern blots also demonstrated the existence of a smaller human transcript (4.5 kb) that 
was similar in size to the small mouse Lyst mRNA, and that appeared to differ from the 
large isoform in distribution of expression in human tissues (Fig. 7). Assuming that the 
genomic derivation of the small human isoform might be the same as the small mouse 
isoform, the 3 '-end of the small human isoform was identified by cloning human intron 
a' using PCR of human genomic DNA with primers derived from exon <j and intron a'. 
Similarly to intron a' in the mouse, the sequence of the 5 '-end of human intron a' 
contained 17 codons in frame with exon a, followed by a stop codon (CHS-ll, inset of 
Fig. 9). cDNA corresponding to this short human isoform was amplified from human 
peripheral blood RNA by RT-PCR with primers from a 5' exon and from intron a', 
indicating that this intron was indeed present in CHS-ll mRNA. 



57 



10 

9 

8 

7- 

t 6 

<n 
z . 

UJ 5 

r- 
5 4 



CHS-l ~13kb 

II ■ ~ 



WD40 



CD 



5' 



Stathmin-like 



•3' 



Probe 2 



CZ3 
Probe 1 



Intron Q ' 



CHS-ll ~6kb 




<5?S! 



Q Q Q Q Q 

Z z Z 

5 5 5 

o o o 



5 5 



glass 

9 B z 5 £ 



115? 
f •; 5 
5 £ t 5 



5 9 T < S- 



t 3 IT UJ 



g 5 ff < 

in "■ C h 



Figure 9. Comparison of the relative abundance of CHS (LYST) mRNA isoforms 
in 50 human tissues after normalization. Probe 2 (specific for the largest isoform CHS-l, 
or LYST-l) and the corresponding signal intensity levels are shown in black. Probe 1 
(identifies all CHS transcripts) and the corresponding signal intensity levels are shown in 
white. Variation of the relative mRNA abundance among tissues was assessed by 
phosphorimaging, dot quantitation, subtraction of background and calculation of the 
intensity percentage for each tissue. The relative mRNA abundance between normalized 
tissues for each probe was expressed as the percentage of the total hybridization signal 
obtained for each tissue with that probe. The average percentage intensity for 
housekeeping genes in all tissues was -2%. No hybridization was evident to control dots, 
containing yeast total RNA, yeast tRNA, E. coli rRNA, E. coli DNA, poly r(A), human 
C tl DNA and human DNA. Exposure time for phosphorimaging was 20 h. 



58 

Nucleotides 1-5905 of human small and long isoform cDNAs are identical, and 
are followed either by intron a' sequence in the short isoform (GenBank accession 
number U84744), or by exon t and the rest of the exons, in the long isoform (inset of Fig. 
9). The predicted intron-encoded amino termini of the short mouse and human isoform 
peptides share 65% identity. 

Additional splicing complexity of smaller isoforms exists. The Lyst-Ill isoform 
lacks exons a and P, while in Lyst-W, exons a, p and y are absent. In both isoforms the 
alternative splicing occurs out of frame and results in termination shortly after the splice 
junction, suggesting that most likely these two isoforms do not have functional 
significance. These splice variants correspond to additional bands observed on northern 
blots (Fig. 7). A schematic representation of the four Lyst isoforms is shown in Fig. 10. 



exons 



LystA, -12 kb 5' 
Lyst-W, -5.9 kb 



co a p 


7 


5 


s 


<j) a 



co | a (3 


Y 


5 


£ 


<|) a 





Lyst-\\\, 720 bp L^ 
Lyst-W/, 744 bp 



* A 

INTRONIC 



*,-' 



CO 



3' 



Figure 10. Schematic representation of the four mRNA isoforms of the Chediak- 
Higashi syndrome gene (*, stop codon; A, poly-A). 



59 

In an effort to evaluate the functional significance of the two largest Lyst 
isoforms, genetic complementation experiments were initiated in collaboration with Dr. 
Stephen Brandt, Vanderbilt University. Full-length Lyst-l and Lyst-ll isoforms were 
amplified from mouse bone marrow cDNA by long-range PCR and cloned in expression 
vectors. The resulting constructs were expressed in cultured fibroblasts from beige mice, 
exhibiting the characteristic giant perinuclear granules in the cytoplasm. The effect was 
evaluated by observing the changes in the size and distribution of specifically stained 
lysosomes and endosomes. Some cells, expressing full-length Lyst-l, demonstrated more 
peripheral distribution of the stained granules in the cytoplasm and slight reduction of 
their size. Such changes were not visible in cells expressing the smaller Lyst-ll isoform, 
supporting the hypothesis that the largest splice variant (Lyst-l) represents the mRNA 
isoform of primary functional significance. However, since the effects of the 
complementation were not very prominent and were not observed in all cells expressing 
Lyst-l, the significance of the described genetic complementation is questionable. 

Analysis of northern blots of mouse mRNA had suggested that the relative 
abundance of the large and small transcripts differed from tissue to tissue (Fig. 7). The 
relative abundance of mRNA isoforms of the homologous gene in human tissues at 
different developmental stages was examined (Fig. 9) by sequential hybridization of a 
poly(A) + RNA dot-blot with a cDNA probe (inset of Fig. 9, Probe 2, shown in black, 
nucleotides 10941-11590 of human CHS, GenBank accession No U67615) specific for 
the largest isoform and with a cDNA probe (inset of Fig. 9, Probe 1, shown in white, 
nucleotides 190-445 of human CHS, GenBank accession No U70064 and U84744) that 
identifies all CHS transcripts (Fig. 9). The inset in Fig. 9 shows the human 5.8 kb cDNA 



60 

isoform (CHS-ll, or LYST-ll, which arises through incomplete splicing, with intron a' 
supplying the 3 '-end of the transcript), the largest isoform (CHS-l, or LYST-I, 13.5 kb, 
which results from removal of intron a') and the location of the probes. The quantity of 
poly(A) + RNA on the blot was normalized to eight housekeeping genes (phospholipase, 
ribosomal protein S9, tubulin, highly basic 23 kDa protein, glyceraldehyde-3 -phosphate 
dehydrogenase, hypoxantine guanine phosphoribosil transferase, P-actin and ubiquitin). 

Using a probe that hybridized only to the largest mRNA isoform on northern 
blots, transcripts were most abundant in thymus (adult and fetal), peripheral blood 
leukocytes, bone marrow and several regions of the adult brain (Fig. 9). Interestingly, the 
largest mRNA isoform was not detected in fetal brain. There was also low relative 
expression of this isoform in heart, lung, kidney and liver in both adult and fetal tissues. 

A somewhat different pattern of relative expression was evident upon 
rehybridization of the blot with a probe derived from the 5 '-end of the coding domain of 
the CHS gene, a region that hybridized to all mRNA isoforms on northern blots. 
Consistent with the transcription pattern of the largest isoform, this probe detected 
abundant expression on peripheral blood leukocytes, thymus (adult and fetal) and bone 
marrow. However, several tissues with abundant large isoform transcripts exhibited 
considerably less relative expression with the 5 '-probe, including most regions of the 
adult brain, fetal and adult thymus, and spleen. Furthermore, several tissues with 
negligible relative transcription of the large isoform exhibited increased relative 
expression with the 5 '-probe, including adult and fetal heart, kidney, liver, and lung; adult 
aorta, thyroid gland, salivary gland and appendix; and fetal brain (Fig. 9). 



61 

Identification and Characterization of Lyst2. a Brain-Specific Member of the Chediak- 

Hi gashi Syndrome Gene Family 

Identification and Sequence Analysis 

In an effort to identify genes similar to the CHS gene, extensive database searches 
were performed. BLASTX comparison of mouse Lyst (U70015) to public EST databases 
revealed a significant, but non-identical, match with an uncharacterized human EST 
(R17955). The corresponding partial-length cDNA clone (#32273, length 1986 bp), 
derived from a human infant brain cDNA library, was sequenced. An additional 250 bp of 
cDNA sequence, located immediately 5' of clone 32273, was obtained by insert-vector 
PCR of a human fetal brain cDNA library. The resulting total sequence (2.2 kb, Fig. 1 la) 
corresponded to the 3' end of the coding domain and the 3' UTR of a novel gene that was 
designated LYST2 (lysosomal trafficking regulator 2, Fig. lib). Amino acid identity of 
LYST2 with LYST varied from region to region: LYST2 amino acids 16-222 shared 50.2 
% identity with LYST residues 3215-3425. This region corresponds to the BEACH 
domain (amino acids 3116-3461) of LYST, and exhibited sequence similarity to 
anonymous ORFs from S. cerevisiae, C. elegans and human CDC4L protein (Nagle et 
al, 1996). In contrast, the WD repeat-containing carboxy-terminal domain of LYST, 
which is predicted to assume a six-bladed beta-propeller structure similar to the 13-subunit 
of heterotrimeric G proteins (Sondek et al., 1996), shared little amino acid similarity with 
LYST2. The latter region of LYST2 did, however, contain LYST-like WD repeats, and 
also exhibited significant sequence similarity to the B-subunit of heterotrimeric G proteins 



62 

(30.4 % identity LYST2 amino acids 368-501 to GB {P49027}). The LYST 3' UTR 
demonstrated 42.1% identity to 584 bp of the 3' UTR of LYST2. 

In an effort to identify the mouse homolog of human LYST2, a mouse embryo 
cDNA library was screened with a human LYST2 probe, and two overlapping clones of 
total length of -2.5 kb were identified (Fig. lie). The corresponding partial mouse 
putative protein (Lyst2, Fig. lid) exhibited 29.4% identity over 676 amino acids to 
mouse Lyst and 96.2 % identity in 557 amino acids to human LYST2. 
Genetic Mapping 

In order to confirm that Lyst and Lyst2 are indeed two different genes, and to 
evaluate the potential candidacy of Lyst2 for mouse coat color mutations, Lyst2 was 
mapped by cross-hybridization in the mouse using unique CAST/EiJ restriction fragment 
length polymorphisms with Mspl (-4.1 kb) and EcoKl (-3.6 kb). Linkage analysis using 
DNA from 93 intersubspecific backcross [C57BL/6J-V X (C57BL/6J-6g >/ x CAST/EiJ) 
F,] mice revealed that Lyst2 maps on mouse Chr 3 (Fig. 12). The best gene order and 
recombination frequency (± standard error) were: centromere - D3MU21 - 6.45±2.5 cM - 
Lyst2 - 5.38±2.3 cM - £>5MfY22-telomere (GenBank accession number AF072372). 

LYST2 was mapped on human Chr 13 by hybridization to Southern blots of 
human-rodent somatic cell hybrids, in collaboration with Dr. Margaret Wallace. No 
cross-hybridization to LYST was observed (Fig. 13). While the CHS gene maps on human 
Chr 1 and on mouse chromosome 13, LYST2 was localized to human Chr 13 and to 
mouse chromosome 3, confirming that LYST and LYST2 are indeed two different genes. 



63 



a) Human LYST2 DNA: 

1 CCGAAGAGAG CTGTGTTTTA TGCAGAGCGT TATGAGACAT GGGAAGATGA 
51 TCAAAGCCCA CCCTACCATT ATAATACCCA TTATTCAACA GCAACATATA 
101 CTTTATCCTG GCTTGTTCGA ATTGAACCTT TCACAACCTT CTTCCTCAAT 
1 5 1 GCAAATGATG GAAAATTTGA TCATCC AGAT CGAACCTTCT CATCCGTTGC 
201 AAGGTCTTGG AGAACTAGTC AGAGGGATAC TTCTGATGTA AAGGAACTAA 
251 TTCCAGAGTT CTACTACCTA CCAGAGATGT TTGTCAACAG TAATGGATAT 
301 AATCTTGGAG TCAGAGAAGA TGAAGTAGTG GTAAATGATG TTGATCTTCC 
351 CCCTTGGGCA AAAAAACCTG AAGACTTTGT GCGGATCAAC AGGATGGCCC 
401 TAGAAAGTGA ATTTGTTTCT TGCCAACTTC ATCAGTGGAT CGACCTTATA 
45 1 TTTGGCTATA AGCAGCGAGG ACCAGAAGCA GTTCGTGCTC TGAATGTTTT 
501 TCACTACTTG ACTTATGAAG GCTCTGTGAA CCTGGATAGT ATCACTGATC 
551 CTGTGCTCAG GGAGGCCATG GAGGCACAGA TACAGAACTT TGGACAGACG 
601 CCATCTCAGT TGCTTATTGA GCCACATCCG CCTCGGAACT CTGCCATGCA 
651 CCTGTGTTTC CTTCCACAGA GTCCGCTCAT GTTTAAAGAT CAGATGCAAC 
701 AGGATGTGAT AATGGTGCTG AAGTTTCCTT CAAATTCTCC AGTAACCCAT 
75 1 GTGGCAGCCA ACACTCTGCC CCACTTGACC ATCCCCGCAG TGGTGACAGT 
801 GACTTGCAGC CGACTCTTTG CAGTGAATAG ATGGCACAAC ACAGTAGGCC 
851 TCAGAGGAGC TCCAGGATAC TCCTTGGATC AAGCCCACCA TCTTCCCATT 
901 GAAATGGATC CATTAATAGC CAATAATTCA GGTGTAAACA AACGGCAGAT 
951 CACAGACCTC GTTGACCAGA GTATACAAAT CAATGCACAT TGTTTTGTGG 
1001 TAACAGCAGA TAATCGCTAT ATTCTTATCT GTGGATTCTG GGATAAGAGC 
1 05 1 TTCAGAGTTT ATACTACAGA AACAGGGAAA TTGACTCAGA TTGTATTTGG 
1101 CCATTGGGAT GTGGTCACTT GCTTGGCCAG GTCCGAGTCA TACATTGGTG 
1151 GGGACTGCTA CATCGTGTCC GGATCTCGAG ATGCC ACCCT GCTGCTCTGG 
1201 TACTGGAGTG GGCGGCACCA TATCATAGGA GACAACCCTA ACAGCAGTGA 
125 1 CTATCCGGCA CCAAGAGCCG TCCTCACAGG CCATGACCAT GAAGTTGTCT 
1301 GTGTTTCTGT CTGTGCAGAA CTTGGGCTTG TTATC AGTGG TGCTAAAGAG 
1351 GGCCCTTGCC TTGTCCACAC CATCACTGGA GATTTGCTGA GAGCCCTTGA 
1401 AGGACCAGAA AACTGCTTAT TCCCACGCTT GATATCTGTC TCCAGCGAAG 
145 1 GCCACTGTAT CATATACTAT GAACGAGGGC GATTCAGTAA TTTCAGCATT 
1501 AATGGGAAAC TTTTGGCTCA AATGGAGATC AATGATTCAA CACGGGCCAT 
1551 TCTCCTGAGC AGTGACGGCC AGAACCTGGT CACCGGAGGG GACAATGGGG 
1601 TAGTAGAGGT CTGGCAGGCC TGTGACTTCA AGCAACTGTA CATTTAACCC 
1651 TGGATGTGAT GCTGGCATTA GAGCAATGGA CTTGTCCCAT GACCAGAGGA 
1 701 CTCTGATCAC TGGCATGGCT TCTGGTAGCA TTGTAGCTTT TAATATAGAT 
1751 TTTAATCGGT GGCATTATGA GCATC AGAAC AGATACTGAA GATAAAGGAA 
1 801 GAACCAAAAG CCAAGTTAAA GCTGAGGGCA CAAGTGCTGCATGGAAAGGC 
1851 AATATCTCTG GTGGAAAAAA TTCGTCTAC A TCGACCTCCG TTTGTACATT 
1901 CCATCACACC CAGCAATAGC TGTACATTGT AGTCAGCAAC CATTTTACTT 
195 1 TGTGTGTTTT TTCACGACTG AACACCAGCT GCTATCAAGC AAGCTTATAT 
2001 CATGTAAATT ATATGAATTA GGAGATGTTT TGGTAATTAT TTCATATATT 
205 1 GTTGTTTATT GAGAAAAGGT TGTAGGATGT GTCACAAGAG ACTTTTGACA 
2101 ATTCTGAGGA ACCTTGTGTC CAGTTGTTAC AAAGTTTAAG CTTTGAACCT 
2151 AACCTGCATC CCATTTCCAG CCTCTTTTC A AGCTGAGAAA AAAAAAAAAA 
2201 AAAAA 

b) Human LYST2 peptide: 

1 PKRAVFYAER YETWEDDQSP PYHYNTHYST ATYTLSWLVR IEPFTTFFLN 
51 ANDGKFDHPD RTFSSVARSW RTSQRDTSDV K.EL1PEFYYL PEMFVNSNGY 
101 NLGVREDEW VNDVDLPPWA KKPEDFVRIN RMALESEFVS CQLHQWIDLI 
1 5 1 FGYKQRGPEA VRALNVFHYL TYEGS VNLDS ITDPVLREAM EAQIQNFGQT 
201 PSQLLIEPHP PRNSAMHLCF LPQSPLMFKD QMQQDVIMVL KFPSNSPVTH 
251 VAANTLPHLT 1PAWTVTCS RLFAVNRWHN TVGLRGAPGY SLDQAHHLPI 
301 EMDPLIANNS GVNKRQITDL VDQSIQINAH CFVVTADNRY ILICGFWDKS 
351 FRVYTTETGK LTQIVFGHWD WTCLARSES YIGGDCYIVS GSRDATLLLW 
401 YWSGRHH1IG DNPNSSDYPA PRAVLTGHDH EWCVSVCAE LGLVISGAKE 
45 1 GPCLVHTITG DLLRALEGPE NCLFPRLISV SSEGHCIIYY ERGRFSNFSI 
501 NGKLLAQMEI NDSTRAILLS SDGQNLVTGG DNGVVEVWQA CDFKQLYI* 

c) Mouse Lyst2 DNA: 

1 GCAGCAGGGC GAACCGGACC TCTGTGATGT TTAATTTTCC TGACCAAGCA 
51 ACAGTTAAAA AAGTTGTCTA CAGCTTGCCT CGGGTTGGAG TGGGGACCAG 
101 CTATGGTTTG CCACAAGCCA GGAGGATATC ACTGGCCACT CCTCGACAGC 
1 5 1 TGTATAAGTC TTCCAATATG ACTCAGCGCT GGCAAAGAAG GGAAATCTCC 
20 1 AACTTTGAGT ATTTGATGTT TCTCAACACG ATAGCAGGTC GGACGTATAA 
25 1 TGATCTGAAC CAGTATCCTG TGTTTCCATG GGTGTTAACA AACTATGAAT 



64 



301 CAGAGGAGTT GGACCTGACT CTCCCAGGAA ACTTCAGGCA TCTGTCAAAG 

351 CCAAAAGGTG CTTTGAACCC GAAGAGAGCA GTGTTTTACG CAGAGCGCTA 

401 TGAGACATGG GAGGAGGATC AAAGCCCACC CTTCCACTAC AACACACATT 

451 ACTCAACGGC GACTTCCCCC CTTTCATGGC TTGTTCGGAT TGAGCCATTC 

501 ACAACCTTCT TCCTCAATGC AAATGATGGG AAATTTGACC ATCCAGACCG 

55 1 AACCTTCTCA TCCATTGCAA GGTCATGGAG AACCAGTCAG AGAGATACAT 

601 CCGATGTCAA GGAACTAATT CCAGAGTTCT ATTACGTACC AGAGATGTTT 

65 1 GTCAACAGCA ATGGGTACCA TCTTGGAGTG AGGGAGGACG AAGTGGTGGT 

701 TAATGATGTG GACCTGCCCC CCTGGGCCAA GAAGCCAGAA GACTTTGTGC 

751 GGATCAACAG GATGGCCCTG GAAAGTGAAT TTGTTTCTTG CCAACTCCAT 

801 CAATGGATTG ACCTTATATT TGGCTACAAA CAGCGAGGGC CAGAGGCAGT 

851 CCGTGCTCTC AATGTTTTCC ACTACTTGAC CTACGAAGGC TCTGTAAACC 

901 TGGACAGCAT CACAGACCCT GTGCTCCGGG AGGCCATGGT TGCACAGATA 

951 CAGAACTTTG CCCAGACGCC ATCTCAGTTG CTCATTGAGC CGCATCCGCC 

1001 TAGGACTTCA GCCATGCATC TGTGTTCCCT TCCACAGAGC CCACTCATGT 

1051 TCAAAGATCA GATGCAGCAG GATGTGATCA TGGTGCTGAA GTTTCCATCC 

1101 AATTCTCCTG TGACTCATGT GGCTGCCAAC ACCCTGCCCC ACCTGACCAT 

1151 CCCTGCAGTG GTGACAGTGA CCTGCAGCCG ACTGTTTGCA GTGAACAGAT 

1201 GGCACAACAC AGTCGGCCTC AGAGGAGCCC CCGGATACTC CTTGGATCAA 

125 1 GCACACCATC TTCCCATTGA GATGGACCCA TTAATCGCAA ATAACTCTGG 

1301 TGTGAACAAG CGGCAGATCA CAGACCTTGT AGACCAGAGC ATCCAGATCA 

1351 ATGCCCACTG CTTCGTGGTC ACAGCTGATA ATCGCTACAT CCTCATCTGT 

1401 GGGTTTTGGG ATAAAAGTTT CAGAGTTTAC TCGACAGAAA CAGGGAAACT 

1451 GACACAGATT GTATTTGGCC ACTGGGATGT TGTCACATGC CTGGCCAGGT 

1501 CGGAGTCCTA CATTGGTGGA GACTGCTACA TAGTGTCTGG ATCTCGGGAC 

1551 GCCACCTTGC TTCTCTGGTA CTGGAGTGGG CGTCACCACA TCATCGGAGA 

1601 CAACCCCAAT AGCAGTGACT ATCCTGCGCC CAGAGCTGTC CTCACAGGCC 

1651 ATGACCATGA AGTTGTCTGT GTCTCCGTCT GTGCAGAACT CGGACTCGTT 

1701 ATCAGTGGTG CTAAAGAGGG CCCTTGCCTC GTTCATACCA TCACTGGAAA 

1751 TCTGCTGAAG GCCCTGGAAG GACCAGAAAA CTGCTTATTT CCACGCCTAA 

1 801 TTTCGGTATC CAGTGAAGGC CACTGCATCA TATATTATGA GCGAGGACGG 

1851 TTTAGC AACT TCAGCATCAA TGGGAAACTT TTGGCTCAAA TGGAGATCAA 

1 90 1 TGATTCCACT AGGGCTATTC TCCTGAGCAG CGATGGACAG AACCTGGTGA 

1951 CTGGAGGGGA CAATGGTGTG GTGGAGGTCT GGCAGGCCTG TGACTTTAAG 
2001 CAGCTGTACA TTTACCCAGG ATGTGATGCT GGCATTAGAG CGATGGATTT 
2051 ATCCCATGAC CAAAGGACTC TGATCACTGG CATGGCTTCC GGCAGCATTG 
2101 TACTTTTAAT ATAGATTTTA ATCGGTGGCA TTATGAGC AT CAGAACAGTA 
2151 CTGAAGAGAA GCAGCAGAAG CCACATTCAA GTGAGAGCAC AAGTGCTTCT 
2201 GTGGAAAGGC AGTATCTCTG GTGGGACGCT GGTCCACATC GGCCTCTGCT 
2251 TGTACATCCA TCCCACCCAG CAGTCGCCGA ACATCATAGT CGGGAGCCAT 
2301 TTCACCCTGT TTTTCCAGGA CTGAACACCA GCTGCTGTCA AGCAAGCTTA 
235 1 TATCATGTAA ATTATCTGAA TTAGGAGCCG TTTTGGTAAT TATTTCATAT 
2401 ATCGCCGTTT ATTGAGAAAA GGTTGTAGGA AGCCTCACAA GAGACTTTTG 
245 1 ACAATTCTGA GGAACCTTGT GCCCAGTTGT TACAAAGTTT AAGCTTTGAA 
CCTAACTTGC ATCCCATTTC CAGCCTCGGG CTTCACTCGT GCC 

d) Mouse Lyst2 peptide: 

1 SRANRTSVMF NFPDQATVKK VVYSLPRVGV GTSYGLPQAR R1SLATPRQL 
5 1 YKSSNMTQRW QRRE1SNFEY LMFLNTIAGR TYNDLNQYPV FPWVLTNYES 
101 EELDLTLPGN FRHLSKPKGA LNPKRAVFYA ERYETWEEDQ SPPFHYNTHY 
1 5 1 STATSPLSWL VRIEPFTTFF LNANDGKFDH PDRTFSSIAR SWRTSQRDTS 
201 DVKELIPEFY YVPEMFVNSN GYHLGVREDE WVNDVDLPP WAKKPEDFVR 
251 INRMALESEF VSCQLHQWID LIFGYKQRGP EAVRALNVFH YLTYEGSVNL 
301 DSITDPVLRE AMVAQIQNFA QTPSQLLIEP HPPRTSAMHL CSLPQSPLMF 
351 KDQMQQDVIM VLKFPSNSPV THVAANTLPH LTIPAWTVT CSRLFAVNRW 
401 HNTVGLRGAP GYSLDQAHHL PIEMDPLIAN NSGVNKRQIT DLVDQSIQIN 
451 AHCFWTADN RYILICGFWD KSFRVYSTET GKLTQIVFGH WDVVTCLARS 
501 ESY1GGDCYI VSGSRDATLL LWYWSGRHHI IGDNPNSSDY PAPRAVLTGH 
551 DHEWCVSVC AELGLVISGA KEGPCLVHTI TGNLLKALEG PENCLFPRLI 
601 SVSSEGHCII YYERGRFSNF SINGKLLAQM E1NDSTRAIL LSSDGQNLVT 
651 GGDNGWEVW QACDFKQLY1 YPGCDAGIRA MDLSHDQRTL ITGMASGSIV 
LLI* 

Fig. 1 1 . Partial cDNA (a, c) and predicted amino acid (b, d) sequences of human 
(a, b) and mouse (c, d) lysosomal trafficking regulator 2, LYST2. 



65 



a 

bbbc 

"•4 — B 




D3Mit21\ 
Lyst2 j 

D3Mit22\ 



DBDBD 

DniiD 

DD1DB 



#ofmice37 45 4 2 4 1 



C 

D3MU21 

Lyst2 
D3Mit22 



~cM 19.2 

6.45±2.5 cM 
5.38+2.3 cM 

~cM 33.7 



£coKI 



Figure 12. Genetic mapping of Lyst2 on mouse chromosome 3. 

a) Autoradiograph of a Southern blot of mouse genomic DNA, digested with 
EcoRI and hybridized to &Lyst2 probe. Molecular size standards (in kilobases) are shown 
to the left. The arrowhead indicates the RFLP, which was used for the genetic mapping 
and was present in DNA from (C57BU6}-bg f x CAST/EU) Fj (be) but not in 
homozygous C57BU6J-bg' (bb) DNA. 

b) Haplotype analysis of Lyst2, D3MU21 and D3MU22 on mouse chromosome 3 
in 93 [C57BL/6J-^ X (C57BL/6J-^ x C AST/EiJ) Fij intersubspecific backcross mice. 
Black squares represent the homozygous C57BL/6J pattern and white squares, the Fi 
partem. The number of mice of each haplotype is indicated. 

c) Composite genetic map of part of mouse chromosome 3, showing the relative 
position of the Lyst2 locus. Marker locations are expressed as genetic distance from the 
centromere (in centimorgans), and intermarker distances (± standard error) are shown. 



66 



r- CD 

E I E 

X ^ 22 13 14 15 16 17 I 

9.4 — 

6.6 — 




Figure 13. Mapping of LYST2 on human chromosome 13 by human-rodent 
somatic cell hybrids. An EcoRl monochromosomal somatic cell hybrid Southern blot was 
hybridized to [a- 32 P]dCTP-labeled LYST2 probe. Lane 1 contains control human DNA; 
lane 2 - control mouse DNA; lane 3 - human Chr 22 on hamster background; lanes 4 to 8 
- human Chr 13, 14, 15, 16, and 17, respectively, on mouse background; lane 9 - control 
hamster DNA. Molecular size standards (in kilobases) are shown to the left of the gel. 



67 

Expression Analysis 

In order to determine the size of the LYST2 transcript(s) and their abundance in 
different organs, expression analysis was performed using northern blots from various 
human and mouse tissues. 

Transcript size in mouse and human. Hybridization of a LYST2 probe to northern 
blots of mouse and human tissues (Fig. 14) revealed that the largest band was -12-13 kb, 
very similar to the size of the largest isoform (LYST-l) of the Chediak-Higashi syndrome 
gene (Barbosa et al, 1997). Low levels of additional transcripts of ~6 kb and ~5 kb were 
visible in mouse brain RNA. 

Transcript abundance in mouse. Hybridization to poly(A) + northern blots from 8 
mouse tissues showed that Lyst2 was abundantly expressed only in brain. Moderate 
expression was observed in kidney, and weak expression - in heart, lung, skeletal muscle 
and testis. The gene was not expressed in spleen and liver (Fig. 14a). 

Transcript abundance in human. In northern blots of selected human tumor cell 
lines (Fig. 14b), LYST2 was moderately expressed in melanoma and colorectal 
adenocarcinoma, weakly expressed in HeLa, lymphoblastic leukaemia and Burkitt's 
lymphoma cells and not expressed in promyelocytic and myelogenous leukaemia, or in 
lung carcinoma lines. In 7 normal human tissues, LYST2 was weakly expressed in spleen, 
lymph node, thymus and appendix and not expressed in peripheral blood leukocyte, bone 
marrow and fetal liver (Fig. 14c). 

The relative abundance of LYST and LYST2 mRNA in 50 human tissues was 
compared by sequential hybridization of corresponding gene-specific probes to a 



68 

poly(A) + RNA dot blot (Fig. 1 5). The quantity of poly(A) + RNA loaded on the blot was 
normalized to 8 housekeeping genes (phospholipase, ribosomal protein S9, tubulin, a 
highly basic 23 kDa protein, glyceraldehyde-3 -phosphate dehydrogenase, hypoxanthine 
guanine phosphoribosil transferase, B-actin and ubiquitin). 

When a probe specific for the largest mRNA isoform of LYST was hybridized to 
the blot, transcripts were most abundant in immune system organs (adult and fetal 
thymus, peripheral blood leukocytes, bone marrow) and in several regions of adult brain 
(Fig. 15). There was moderate to low relative LYST expression in most of the other 
tissues tested. Interestingly, no LYST expression was detected in fetal brain, as well as in 
salivary gland, kidney, lung and fetal heart. 

Following hybridization of the same blot with a Z,y5T2-specific probe, a different 
pattern of expression was observed (Fig. 15). LYST2 transcripts were most abundant in 
whole brain (adult and fetal) and in 14 specific adult brain regions, as well as in kidney, 
while there was low to negligible LYST2 expression in the rest of the tissues. 



69 



a 



12 3 4 5678 



12 3 4 5 6 7 8 



12 3 4 5 6 7 




9.5- 
7.5- 

4.4- 

2.5- 

1.4- 




Mouse 



Human 



Human 



Figure 14. Northern blot analysis of mouse and human LYST2. 
Northern blots of 2 ug poly(A) + RNA from various mouse tissues (a), human 
cancer cell lines (b), or human lymphoid tissues (c), hybridized to a [a- 32 P]dCTP- 
labeled LYST2 probe. Lanes: (a) 1-heart, 2-brain, 3-spleen, 4-lung, 5-liver, 6- 
muscle, 7-kidney, 8-testis; (b) 1 -promyelocytic leukemia, 2-HeLa, 3- 
myelogenous leukemia, 4-lymphoblastic leukemia, 5-Burkitt's lymphoma, 6- 
adenocarcinoma, 7-lung carcinoma, 8-melanoma; (c) 1 -spleen, 2-lymph node, 3- 
thymus, 4-appendix, 5-leukocyte, 6-bone marrow, 7-fetal liver. Molecular size 
standards (in kb) are shown to the left. 



70 



10 



9 










b^kst 


8 


1 ! 




□ LES72 


7 

t 6 

CO 

Z 5 n n 

1- 

z 

* 4 

3 n 








2 1 


Ill ril 








;U | 


I. J 1ljl.1i 


HL...J....L1 


lllj ,Jln 


L I till ll 



< Ul 

1 



< O 5 S O 



Q fc < UJ Z CC 






§ i 

ai j 

2 H 

3 8 



2 3 g 

E g 2 1 



is 



i a 



Q Q > OC 



a a i t & is i 



I j 



5 * £ | S 

-J U UJ £ UJ 



Ell 



3 i 



w Q _i 

5 ; « 



I | 



Figure 15. Comparison of the relative abundance of ZKSTand IKSTi transcripts 
in 50 human tissues after normalization. Variation of the relative mRNA abundance 
among tissues was assessed by phosphorimaging, dot quantitation, subtraction of 
background and calculation of the intensity percentage for each tissue. The relative 
mRNA abundance between normalized tissues for each probe was expressed as the 
percentage of the total hybridization signal obtained for each tissue with that probe. The 
average percentage intensity for housekeeping genes in all tissues was -2%. No 
hybridization was evident to control dots, containing yeast total RNA, yeast tRNA, E. 
coli rRNA, E. coli DNA, poly r(A), human C tl DNA and human DNA. Exposure time 
for phosphorimaging was 20 h. 



71 

Identification of Proteins that Interact with the CHS Protein and with LYST2. Using a 

Yeast Two-Hybrid Approach 

In an effort to investigate the function of the LYST and LYST2 proteins, two 
types of screens for protein-protein interactions were performed by the yeast two-hybrid 
method, as described in MATERIALS AND METHODS. In a "forward" screen, one 
hybrid consisted of the DNA binding domain of the yeast transcriptional activator Gal4 
fused to a "bait" portion of LYST or LYST2. The other hybrid consisted of the Gal4 
activation domain fused to "prey" sequences encoded by a mammalian cDNA library. In 
a "reverse" screen, the "bait" part of LYST or LYST2 was fused to the Gal4 activation 
domain, and the "prey" sequences of the mammalian cDNA library were fused to the 
Gal4 DNA binding domain. The activation domain vector contained the LEU2 gene for 
selection in yeast deficient in leucine biosynthesis, and the DNA-binding domain cloning 
vector included the TRP1 gene for selection in yeast deficient in tryptophane 
biosynthesis. Each of the vectors was transformed into complementary (a and a) mating 
types of yeast. Mating was carried out to express both vector constructs within the same 
yeast cells, thus allowing interaction to occur. Interaction between the bait and prey 
proteins led to transcriptional activation of reporter genes containing cis-binding elements 
for Gal4. The reporter genes encoding the indicator protein beta-galactosidase, and 
metabolic markers for uracil and histidine auxotrophy, were included in specific fashion 
in one or the other of the yeast strains used in the mating, as described in the 
MATERIALS AND METHODS chapter. As a result, yeast were selected for expression 
of both fusion constructs, for successful mating, and for expression of LYST- or LYST2- 



72 

interacting proteins (IPs). Yeast clones that contained interacting proteins were grown in 
individual wells of microtiter plates and the plasmids containing the IP-sequences were 
isolated and characterized. Numerous tests (for bait self-activation, prey self-activation, 
non-specific binding, sequencing of all baits, matrix mating, (3-galactosidase tests for 
haploids and for diploids) were performed, as described in the MATERIALS AND 
METHODS chapter, to determine the specificity of the detected baitprey interactions. 
The following interactions, summarized in Table 5, passed all confirmation tests. 

In the forward screens, four different molecules were found to interact with the 
CHS protein. LYST fragment 190-1056 interacted with sequences identical to the 14-3-3 
protein. Three independent isolates of 14-3-3 sequence, starting at nucleotides 270, 306, 
and 330, representing the same region of the molecule (as shown in APPENDIX, 
Sequence 1), were identified. Another sequence which was found to interact with LYST 
fragment 3190-4032 was identical to casein kinase II P-subunit. Four independent isolates 
of CKII-p, starting at nucleotides 1, 4, 31, and 82 (APPENDIX, Sequence 3) and 
representing the same region of the molecule, were identified. LYST fragment 6586-7449 
was found to interact with a sequence identical to human hepatocyte growth factor- 
regulated tyrosine kinase substrate (Hrs), starting at nucleotide 326 (APPENDIX, 
Sequence 4). Another identified sequence interacting with LYST fragment 9037-9585 
was identical to the EST cg51287.dl0 sequence (IP-11, APPENDIX, Sequence 23), 
starting at nucleotide 1 . 

In the reverse screens, a total of 22 molecules were identified as LYST- or 
LYST2-IPs. Ten of those were identical to published proteins and thirteen molecules 



73 

were identified as EST sequences (eleven novel and two known ESTs). Identified 
sequences interacting with the LYST fragment 190-1056 were identical to 14-3-3 protein 
sequence starting at nucleotides 270, 288 and 354, i.e. representing the same domain of 
14-3-3 protein identified in the forward screen (APPENDIX, Sequence 1), to B 14-3-3 
protein sequence starting at nucleotide 466 (APPENDIX, Sequence 2), to calmodulin 
sequence starting at nucleotide 474 (APPENDIX, Sequence 5), to estrogen receptor- 
related protein, starting at nucleotide 756 (as shown in APPENDIX, Sequence 6), and to 
EST cg50173.dl0 sequence (IP-7, APPENDIX, Sequence 19), starting at nucleotide 1. 
The sequence identical to 14-3-3 protein, starting at nucleotide 270, also interacted with 
the LYST fragment 4009-4821. LYST fragment 6586-7449 was found to interact with 
sequences identical to importin P-subunit (starting at nucleotide 1970, APPENDIX, 
Sequence 7), imogen 38 (starting at nucleotide 492, APPENDIX, Sequence 8), DGS-I (a 
gene with unknown function, isolated from the DiGeorge syndrome critical region, 
starting at nucleotide 455, APPENDIX, Sequence 9), EST cg50136.f6 (IP-1, starting at 
nucleotide 1, APPENDIX, Sequence 3), EST AA0 10799 (IP-2, starting at nucleotide 1, 
APPENDIX, Sequence 4), EST cg50136.a7 (IP-4, starting at nucleotide 1, APPENDIX, 
Sequence 16), EST cg50175.c7 (IP-5, starting at nucleotide 25, APPENDIX, Sequence 
17), EST cg50138.g5 (IP-6, starting at nucleotide 1, APPENDIX, Sequence 18), EST 
cg50175.h7 (IP-8, starting at nucleotide 1, APPENDIX, Sequence 20), EST cg50136.a5.b 
(IP-10, starting at nucleotide 1, APPENDIX, Sequence 22), and EST cg49432.h3 (IP-12, 
starting at nucleotide 1, APPENDIX, Sequence 24). The sequence identical to EST 
cg50138.g5 (IP-6, starting at nucleotide 1) interacted also with LYST fragment 10576- 



74 

11611. Identified sequences interacting with LYST fragment 9502-10590 were identical 
to atrophin-1 (starting at nucleotide 2660, APPENDIX, Sequence 10), embryonic fyn 
substrate 2 (Efs2, starting at nucleotide 1665, APPENDIX, Sequence 1 1), similar to EST 
cg50136.cl0 (IP-3, starting at nucleotide 356, APPENDIX, Sequence 5), identical to EST 
KIAA0192 (IP-9, starting at nucleotide 5092, APPENDIX, Sequence 21), and similar to 
EST cg50138.el.b (IP-13, starting at nucleotide 75, APPENDIX, Sequence 25). The 
sequence identical to EST cg50136.cl0 (IP-3) interacted also with LYST fragment 9037- 
9585. The sequences identical to casein kinase II P-subunit (starting at nucleotides 1 and 
58) interacted not only with the LYST fragment 3190-4032 in the forward screen, but 
also with LYST fragment 10576-1 161 1 in the reverse screen. 

In summary, eleven LYST-interacting sequences were found to be identical to 
published sequences encoding proteins (14-3-3, B 14-3-3, Hrs, calmodulin, casein kinase 
II (3-subunit, Efs2, importin P-subunit, estrogen receptor-related protein, imogen 38, 
atrophin-1, and DGS-I), two LYST-interacting sequences were found to be highly 
homologous or identical to published EST sequences (AA0 10799 and KIAA0192, 
encoding for IP-2 and IP-9, respectively), and eleven LYST-interacting sequences were 
found to be novel ESTs (cg50136.f6, cg50136.cl0, cg50136.a7, cg50175.c7, cg50138.g5, 
cg50173.dl0, cg50175.h7, cg50136.a5.b, cg51287.dl0, cg49432.h3.b, and cg50138.el.b; 
encoding for IP-1, IP-3, IP-4, IP-5, IP-6, IP-7, IP-8, IP-10, IP-11, IP-12, and IP-13 
respectively). 

In the reverse screen searching for LYST2-IPs, one interacting sequence was 
found to be identical to the published sequence encoding human brain factor 2 (HBF-G2, 



75 

starting at nucleotide 1638, APPENDIX, Sequence 2) and one to the same region 

(starting at nucleotide 354, APPENDIX, Sequence 1) of 14-3-3 protein, identified to 

interact with LYST. The nucleic acid sequences and corresponding amino acid sequences 

of all LYST- and LYST2-interacting proteins are shown in APPENDIX, Sequences 1-25. 

A summary of the described protein-protein interactions is shown in Table 5. 

Table 5. Protein interactions identified by forward and reverse yeast two-hybrid 
screens using fragments of LYST and LYST2 as baits 



BAIT FRAGMENT 

AND POSITION, 

bp* 


INTERACTING PROTEINS IDENTIFIED IN 


FORWARD SCREENS 


REVERSE SCREENS 


LYST 190-1056 


14-3-3 protein 


14-3-3 protein; B14-3-3; 
Calmodulin; IP-7; Estrogen 
receptor-related protein 


LYST 3190-4032 


Casein kinase II P-subunit 




LYST 4009-4821 




14-3-3 protein 


LYST 6586-7449 


Hepatocyte growth factor - 
regulated tyrosine kinase 
substrate (Hrs) 


Importin P-subunit; Imogen 38; 
DGS-I (DiGeorge syndrome); 
IP- 1 ,-2,-4,-5,-6,-8,- 1 0,- 1 2 


LYST 9037-9585 


IP- 11 


IP-3 


LYST 9502-10590 




Atrophin-1; Embryonic Fyn 
substrate 2 (Efs2); IP-3, -9,- 13 


LYST 10576-11611 




Casein kinase II P-subunit; IP-6 


LYST2 774-1424 




14-3-3 protein; Human brain 
factor-2 (HBF-G2) 



* According to GenBank accession number U67615 for LYST; for LYST2, fragment 
positions are relative to the beginning of the known sequence 



76 

The potential functional significance of the interactions between LYST/LYST2 
and the previously published proteins will be discussed in the next chapter. A short 
description of all ESTs, detected as LYST-IPs, and their similarity to known sequences 
follows. 
IP-1 (ESTcg50136.f6. APPENDIX. Sequence 13) 

One identified sequence (cg50136.f6, 503 nucleotides), interacting with LYST, 
was 98% identical to nucleotides 1-128, 90% to nucleotides 339-455, and 71% to 
nucleotides 201-445 of the human EST AA452346. This EST of 455 nucleotides was 
initially obtained from a human Soares total fetus (8-9 week) library (Hillier et al, 1997). 
However, EST AA452346 could be not be used for an extension of EST cg50136.f6 in 
either direction. 

EST cg50136.f6 showed 68% nucleotide identity to mouse T-complex protein 
Tcp-10 genes (GenBank accession No. M73509, M73506, M22601, M73505). An open 
reading frame could be translated from nucleotides 3 to 503. The resulting 167 amino 
acids correspond to a c-terminal or core region of a novel protein, which was designated 
IP-1. Amino acids 2-126 of IP-1 show 68% homology to the c-terminal region (amino 
acids 242-365) of mouse Tcp-10 protein (GenBank accession No. X58170) 

Therefore, IP-1 represents a novel protein with homology to mouse Tcp-10 
protein. Interestingly, another LYST-interactant, IP- 10 (see later in this chapter), shows 
homology to the amino-terminal region of Tcp-10. In addition, LYST-interactant IP-1 1 
has a region homologous to ATP-binding proteins and chaperonin Tcp-1. The nucleotide 
and amino acid sequences of IP-1 are shown in APPENDIX, Sequence 13. 



77 

IP-2 (EST AAO 10799. APPENDIX. Sequence 14) 

Three identical sequences (cg50136.a4, cg50136.d6, cg50136.c9), interacting with 
LYST, were 96% identical to nucleotides 1-441 of the human EST AAO 10799. This EST 
of 495 nucleotides was initially obtained from a human Soares fetal heart (19 weeks) 
library (Hillier et ai, 1995). EST AAO 10799 could not be further extended by database 
searches and was used for further analysis. 

The 5' end of this EST was similar to the human 50 S ribosomal protein LI 7. An 
open reading frame could be translated from nucleotides 3 to 494 of EST AA010799. The 
resulting 1 64 amino acids correspond to a c-terminal or core region of a novel protein, 
which was called IP-2. Amino acids 1-107 of IP-2 show 55% homology to amino acids 5- 
117 of bacterial ribosomal protein L17 (GenBank accession No. S07223, M26414, 
L33834, AE00633, D90905). 

Therefore, IP-2 represents a novel human protein with homology to ribosomal 
protein LI 7. The nucleotide and amino acid sequences of IP-2 are shown in APPENDIX, 
Sequence 14. 
IP-3 TEST cg50136.cl0. APPENDIX. Sequence 15) 

Two identical sequences (cg50136.cl0 and cg50136.gl0), interacting with LYST, 
showed significant homology to published ESTs. An extended expressed sequence of 
1198 nucleotides was assembled from nucleotides 1 to 466 of human fetal lung EST 
W40354 (Hillier et al, 1995), nucleotides 57 to 194 from cg50136.cl0 (corresponding to 
nucleotides 345 to 539 in the extended sequence), nucleotides 1 to 382 from human 
endothelial cell EST AA1 86481 (Hillier et al, 1995), corresponding to nucleotides 483 to 



78 

863 in the extended sequence, nucleotides 336 to 446 from human fetal EST AA436726 
(Hillier et al, 1997), corresponding to nucleotides 529 to 974 in the extended sequence, 
and nucleotides 199 to 423 from EST N49053 from a human multiple sclerosis library 
(Hillier et al, 1995), corresponding to nucleotides 777 to 1 198 in the extended sequence. 
The fragment interacting with LYST starts at nucleotide 345. The extended sequence of 
1 198 nucleotides was used for subsequent analysis. 

Nucleotides 213 to 599 of the extended expressed sequence showed 75% identity 
to nucleotides 3589 to 3975 of rat Olf-1/EBF associated Zn finger protein Roaz 
(GenBank accession No. U92564). An open reading frame could be translated from 
nucleotides 1 to 594. These 198 amino acids represent a core or c-terminal region of a 
novel protein, which was designated IP-3. Amino acids 26 to 197 of IP-3 show 84% 
homology to amino acids 1023 to 1185 of the rat Zn finger protein Roaz. Weaker 
homologies (40 to 50%) were seen to different zinc-finger proteins of several species. 
Interestingly, conserved zinc-finger domains exist in other proteins, e.g., protein kinase C 
and Hrs, that may be associated with LYST (see next chapter). 

Therefore, IP-3 represents a novel human protein with homologies to rat Olf- 
1/EBF associated Zn finger protein Roaz. The nucleotide and amino acid sequences of IP- 
3 are shown in APPENDIX, Sequence 15. 
IP-4 TEST cg50136.a7. APPENDIX. Sequenced) 

One identified prey sequence (cg50136.a7, 493 nucleotides), interacting with 
LYST, was 91% identical to human EST AA009453, starting at nucleotide 1. This EST 
of 505 nucleotides was initially obtained from a human Soares fetal heart (19 weeks) 



79 

library (Hillier et al, 1997). EST AA009453 could be extended in the 5' direction with 
nucleotides 57 to 76 of clone cg50136.a7, resulting in a sequence of 524 nucleotides. 

An open reading frame could be translated from nucleotides 2 to 244. These 81 
amino acids correspond to a carboxy-terminal, proline-rich region of a novel protein, 
which was called IP-4. 

Therefore, IP-4 represents a novel, proline-rich protein without homology to 
known proteins. The nucleotide and amino acid sequences of IP-4 are shown in 
APPENDIX, Sequence 16. 
IP-5 TEST cg50175.c7. APPENDIX. Sequence 17) 

One identified prey sequence (cg50175.c7, 548 nucleotides), interacting with 
LYST, was 98% identical to human cDNA clone AA403189, starting at nucleotide 24. 
This EST of 386 nucleotides was initially obtained from a human Soares total fetus (8-9 
week) library (Hillier et al, 1997). EST AA403189 was extended with nucleotides 418 to 
548 of clone 50175x7, resulting in a sequence of 517 nucleotides. This sequence could 
not be extended further by database searches and was used for subsequent analysis. 

An open reading frame could be translated from nucleotides 1 to 288. This 
corresponds to a 96 amino acid protein, which was designated IP-5. Amino acids 10 to 84 
of IP-5 were found to be 48% homologous to Bacillus subtilis ATP-dependent protease 
La (GenBank accession No. X76424 and P37945), to Bacillus subtilis ATP-dependent 
Lon protease (GenBank accession No. Z75208), and to Bacillus subtilis endopeptidase La 
(GenBank accession No. 140421). 



80 

Therefore, IP-5 represents a novel protein with homology to ATP-dependent 
proteases Lon/La. The nucleotide and amino acid sequences of IP-5 are shown in 
APPENDIX, Sequence 17. 
IP-6 TEST cg50138.g5. APPENDIX. Sequence 18) 

Two identified sequences (cg50138.g5, 498 nucleotides, and the identical 
cg50138.f2), interacting with LYST, showed significant homology to published 
expressed sequences. Nucleotides 222 to 492 of cg50138.g5 were 98% identical to 
nucleotides 1 to 270 of EST AA460131 (588 nucleotides; Hillier et al, 1997). This 
sequence was obtained from a human total fetus (9 weeks) cDNA library. A longer 
sequence was assembled from nucleotides 1-449 of cg50138.g5 and nucleotides 228-588 
of AA460131, and the extended sequence of 753 nucleotides was used for subsequent 
analysis. 

The extended sequence showed homology to several viral nonstructural proteins: 
nucleotides 2 to 487 showed 83% identity to nucleotides 170 to 653 of the nonstructural 
protein Ns2-3 of a border disease virus strain (pestivirus type 3) (GenBank accession No. 
U43603), nucleotides 42 to 405 showed 90% identity to nucleotides 17 to 378 of proteins 
p54, p80 and pl25 of the bovine viral diarrhea virus (GenBank accession No. Z54331). 
Nucleotides 311 to 753 showed 58% identity to nucleotides 3025 to 3464 of the human 
growth hormone and chorionic somatomammotropin genes (GenBank accession No. 
J03071). 

An open reading frame could be translated from nucleotides 1 to 582. These 194 
amino acids represent a core or c-terminal region of a novel protein, which was called IP- 



81 

6. Amino acids 6 to 144 of IP-6 show 93% homology to amino acids 64 to 198 of the 
viral (pestivirus type 3) nonstructural protein Ns2-3 (GenBank accession No. U43603). 
Amino acids 31 to 135 are 98% homologous to amino acids 22 to 126 of the 
nonstructural protein pi 25 of the bovine viral diarrhea virus (GenBank accession No. 
Z54332). BLOCKS analysis searching for similarities to known protein families showed 
some homology to 14-3-3 proteins (amino acids 17 to 68; identities are shown in capital 
letters): Efl SK1QD dLKea mntmm CSRcQ GkhRr Femdr Epksa RycAE cnrlh pAE. 

Therefore, IP-6 represents a novel human protein with homology to viral 
nonstructural proteins. The nucleotide and amino acid sequences of IP-6 are shown in 
APPENDIX, Sequence 18. 
IP-7 TEST cg50173.dl0. APPENDIX. Sequence 19) 

One identified sequence (cg50173.dl0, 449 nucleotides), interacting with LYST, 
showed homology to published expressed sequences. Nucleotides 79 to 325 of 
cg50173.dl0 were 91% identical to nucleotides 30 to 283 of the expressed sequence 
H62553 (293 nucleotides; Hillier et al, 1995). This sequence was obtained from a human 
male fetal (20 weeks) liver and spleen cDNA library. The expressed sequence 
cg50175.dl0 could not be further extended by database searches and was used for further 
analysis. 

An open reading frame could be translated from nucleotides 17 to 289, starting 
with a methionine start codon. These 91 amino acids correspond to a novel protein with a 
calculated molecular weight of 10589,9, which was designated IP-7. Both the nucleotide 
and the amino acid sequence of IP-7 show some homology to Alu-domains. 



82 

Therefore, IP-7 represents a novel human protein without homology to known 
proteins. The nucleotide and amino acid sequences of IP-7 are shown in APPENDIX, 
Sequence 19. 
IP-8 TEST cg50175.h7. APPENDIX. Sequence 201 

One identified sequence (cg50175.h7), interacting with LYST, showed significant 
homology to published expressed sequences. Nucleotides 254 to 555 of cg50175.h7 were 
96% identical to nucleotides 1 to 330 of the expressed sequence T09146. This sequence 
was obtained from a human infant brain cDNA library (Adams et al., 1993). Nucleotides 
61 to 433 of cg50175.h7 were assembled to nucleotides 181 to 330 of T09146, resulting 
in an extended sequence of 523 nucleotides. This sequence could not be further extended 
by database searches and was used for further analysis. 

Nucleotides 2 to 523 of the assembled expressed sequence were 89% homologous 
to the rat mRNA for norbin (GenBank accession No. AB006461; Shinozaki et al, 1997). 
Norbin is a novel brain gene, induced by treatment of tetraethylammonium in rat 
hippocampal slice and accompanied with neurite-outgrowth in neuro 2a cells. The 
neurite-outgrowth-related protein norbin may play a role in neural plasticity because of 
the formation of new synapses. 

An open reading frame could be translated from nucleotides 2 to 523. These 174 
amino acids correspond to a c-terminal or core region of a novel protein, which was 
called IP-8 and which was 98% identical to amino acids 486 to 659 of rat norbin brain 
protein (729 amino acids). 



83 

Therefore, IP-8 represents a novel protein that appears to be the human homolog 
of the rat brain gene norbin. The nucleotide and amino acid sequences of IP-8 are shown 
in APPENDIX, Sequence 20. 
IP-9 TEST KIAA0192. APPENDIX. Sequence 21) 

One identified prey sequence of 499 nucleotides interacting with LYST was 96% 
identical to human mRNA clone D83783 for the KIAA0192 gene, starting at nucleotide 
5092. The mRNA for KIAA0192 was initially obtained from a human male myeloblast 
cell line (Nagase et al, 1996). 

An open reading frame without a methionine start codon could be translated from 
nucleotides 1 to 6372. This sequence represents the carboxy-terminal part of a protein 
which was designated IP-9. 

Since no homologies to known proteins could be identified, it appears that IP-9, 
encoded by the human mRNA for the KIAA0192 gene, represents a novel protein that 
interacts with LYST. The nucleotide and amino acid sequences of IP-9 are shown in 
APPENDIX, Sequence 21. 
IP- 10 TEST cg50136.a5.h. APPENDIX. Sequence 22) 

Two identified sequences (cg50136a5 and cg50136c4), interacting with LYST, 
showed no significant homology to published sequences. Nucleotides 62-309 were 57% 
homologous to nucleotides 61 to 310 of the human immunodeficiency virus envelope 
protein (GenBank accession No. L08337). The interacting sequence of 451 nucleotides 
was used for further analysis. 



84 

An open reading frame could be translated from nucleotides 2 to 451. These 150 
amino acids correspond to a c-terminal or core region of a novel protein, which was 
called IP-10. Amino acids 59 to 139 of IP-10 showed 59% homology to amino acids 29- 
109 of the human T-complex protein TCP-10 (GenBank accession No. U03399). Other 
homologies (-50%) were found to several cytoskeletal proteins (e.g. myosin I isoform, 
kinesin precursor, cytokeratin, caldesmon, NUF1, tropomyosin, neurofilament protein, 
kinesin-like protein KIF1, myosin like protein MLP1, troponin T) and to proteins 
involved in vesicular transport (intracellular protein transport protein, nuclear fusion 
protein Bikl, synaptonemal complex protein). BLOCKS analysis showed homologies to 
postsynaptic proteins, gas vesicles protein, clusterin proteins, clathrin light chain proteins, 
tropomyosin protein and intermediate filament protein. 

Therefore, IP-10 represents a novel protein with homologies to the amino- 
terminal region of Tcp-10 and with homologies to cytoskeletal and vesicular transport 
proteins. It is interesting that the LYST-interactant IP-1 shows homology to the c- 
terminal region of Tcp-10. Furthermore, LYST-interactant IP-1 1 has homologous 
domains to ATP-binding proteins and chaperonin Tcp-1. The nucleotide and amino acid 
sequences of IP-10 are shown in APPENDIX, Sequence 22. 
IP-1 J TEST cg51287.d!0. APPENDIX. Sequence 23^1 

One identified sequence (cg51287.dl0), interacting with LYST, was 90% 
identical to nucleotides 1-325 of the human EST AA3 10287. This EST of 470 
nucleotides was initially obtained from a human Jurkat T-cells library (Adams et al, 
1995). The 5' end of EST AA3 10287 could be extended with nucleotides 154 to 180 of 



85 

EST cg51287.dl0, resulting in a sequence of 497 nucleotides, which was used for further 
analysis. 

An open reading frame could be translated from nucleotides 3 to 497. These 165 
amino acids correspond to a c-terminal or core region of a novel protein, which was 
designated IP-11. No significant homologies could be found using the BLASTN search 
program. Using the BLOCKS analysis program, homologies to ATP-binding proteins and 
chaperonins TCP-1 proteins could be found. 

Therefore, IP-11 represents a novel protein with homologies to ATP-binding 
proteins and TCP-1. The nucleotide and amino acid sequences of IP-11 are shown in 
APPENDIX, Sequence 23. 
IP-12 (ESI cg49432.h3.h. APPENDIX. Sequence 24) 

Two identified sequences (cg50136c6 and cg50136d2), interacting with LYST, 
showed no significant homologies to published sequences. The interacting sequence of 
402 nucleotides was used for further analysis. 

Nucleotides 27-266 of EST cg49432.h3.b were 70% homologous to nucleotides 
1 1 15 to 1348 of the human RNA for the cellular oncogene c-fes (GenBank accession No. 
X52192). 

An open reading frame could be translated from nucleotides 2 to 400. These 133 
amino acids correspond to a c-terminal or core region of a novel protein, which was 
called IP-12. Only weak homologies to known proteins could be found, especially to 
proline-rich regions of different proteins. For example, the proline rich region of IP-12 
consisting of amino acids 57 to 89 was 69% homologous to the TGF-p binding protein 3, 



86 

amino acids 57 to 75 were 66% homologous to a region in human semaphorin IV, 64% 
homologous to StpC139 (Saimiriine herpesvirus 2), 63% homologous to a collagen-like 
protein and 55% homologous to homeobox protein HOX-A4. Although the functions of 
proline-rich regions are often unclear, they could be involved in protein-protein 
interactions. Some transcription factors contain a proline-rich sequence in the 
transcriptional activation domain, which has no DNA-binding activity but is essential for 
activating transcription (Mitchell and Tjian, 1989). Amino acids 14 to 67 of IP- 12 
showed 53% homology to ORF homolog membrane peroxisomal 70 kDa protein P34230 
(ALDP), involved in adrenoleukodystrophy. 

Therefore, IP- 12 represents a novel protein with a proline-rich region. Other 
LYST interactants, IP-4 and Hrs, have also proline-rich regions. The nucleotide and 
amino acid sequences of IP- 12 are shown in APPENDIX, Sequence 24. 
IP- 13 TEST cg50175.cll. APPENDIX. Sequence 25) 

One identified sequence (cg50175.cll, 481 nucleotides), interacting with LYST, 
showed significant homology to published expressed sequences. Nucleotides 1 to 355 of 
cg50175.cll were 96% identical to nucleotides 74 to 428 of the expressed sequence 
H51347 (428 nucleotides; Hillier et al, 1995), obtained from a mouse brain cDNA 
library. The expressed sequence cg50175.cll was extended in the 5' direction with 
nucleotides 1-73 of H51347. This extended sequence showed 96% identity to an 
expressed sequence Hs21767 of the same length. The extended sequence of 554 
nucleotides was used for further analysis. 



87 

The extended expressed sequence of 554 nucleotides showed 71% identity to 
nucleotides 463 to 982 of Xenopus laevis elav-type ribonucleoprotein etr-1 (GenBank 
accession No. U 16800). An open reading frame could be translated from nucleotides 3 to 
554. These 184 amino acids represent the core or c-terminus of a novel protein, which 
was designated IP-13. The amino acid sequence of IP- 13 shows homologies to 
ribonucleoproteins of different species, including 67% homology to Xenopus laevis elav- 
type ribonucleoprotein etr-1 (GenBank accession No. U16800) and 54% homology to the 
human etr-3 protein (GenBank accession No. U69546). 

Therefore, IP-13 represents a novel human protein with homology to 
ribonucleoproteins. The nucleotide and amino acid sequences of IP-13 are shown in 
APPENDIX, Sequence 25. 



DISCUSSION 



Since the predicted mouse and human CHS proteins were novel and unlike any of 
the molecules previously implicated in vesicular transport, their identification did not 
prove immediately helpful in establishing the precise mechanism whereby CHS 
dysregulates protein transport and lysosomal trafficking. Therefore, the main goal of this 
dissertation was to characterize the Chediak-Higashi gene and its products in more detail 
by using functional genomics approaches: 

identifying alternatively spliced Lyst mRNA isoforms and therefore resolving 
the discrepancy between the initially reported Lyst sequences 
identification of mutations in Chediak-Higashi patients and beige alleles 
- identification of proteins that interact with LYST 



Identification and Characterization of Lyst mRNA Isoforms 

In order to investigate the expression of the CHS gene and to explain the 
discrepancy between the previously published Lyst sequences, extensive northern and 
RT-PCR analyses were performed. We demonstrated that each of the previously reported 
bg gene sequences (Barbosa et ai, 1996; Perou et ai, 1996b) is derived from a single 
gene with alternatively spliced mRNAs (Fig. 8). The previously reported sequences are 

88 



89 

derived from non-overlapping parts of two Lyst mRNA isoforms with different predicted 
C-terminal regions. By sequencing RT-PCR products, we have shown that nucleotides 1- 
4706 of Lyst also represent the previously undetermined 5' region of the largest Lyst 
isoform. Alternative splicing at nucleotide 4706 results in bg gene isoforms that contain 
different 3' regions. Splicing from exon a (containing nucleotide 4706) to exon x results 
in an -12 kb mRNA (Lyst-T) that corresponds to the largest band observed on northern 
blots. Incomplete splicing at nucleotide 4706 results in a 5893 bp cDNA (Lyst-ll) that 
contains intron-derived sequence at the 3' end. Lyst-ll corresponds to a smaller mRNA 
observed on northern blots (Barbosa et al., 1996). While several other genes generate an 
alternative C-terminus by incomplete splicing (Myers et al., 1995; Sugimoto et al., 1995; 
Sygiyama et al., 1996; Zhao and Manlley, 1996; Van De Wetering et al., 1996), the bg 
gene is unique in that the predicted structures of the two C-termini are quite different. The 
C-terminus of Lyst-I contains a WD-repeat domain that is similar to the P-subunit of 
heterotrimeric G proteins and which may assume a propeller-like secondary structure 
(Lambright et al., 1996). This domain is absent in Lyst-II. 

Northern blots of human tissues had suggested that transcription of the 
homologous human gene, LYST, had a similar complexity to the mouse. We identified 
two human ESTs homologous to mouse Lyst and described a mutation in one of these in 
a Chediak-Higashi patient (Barbosa et al., 1996). Subsequently, another group published 
the cDNA sequence of the largest LYST isoform, and identified mutations in this gene in 
two additional patients with CHS (Nagle et al, 1996). We have described the 
identification of a second isoform of the human gene. This mRNA encodes a protein of 



90 

1531 amino acids that is homologous to mouse Lyst-ll. Like the latter, this human mRNA 
arises from incomplete splicing and retention of a transcribed intron that encodes the C- 
terminus of the predicted LYST protein. The mouse and human codons unique to this 
short isoform share 65% amino acid identity. The stop codon, however, is not conserved 
precisely between the human and mouse short isoforms. While mouse Lyst-II is predicted 
to contain a C-terminal prenylation motif (CYSP), translation of human short isoform is 
predicted to terminate 22 codons earlier and to lack this motif. 

However, the other predicted structural features of the human short isoform were 
conserved with mouse. The most notable of these was a region similar in sequence to 
stathmin (amino acids 376-540) (Barbosa et al, 1996). While the mouse and human CHS 
genes had an overall amino acid identity of 81%, identity in the stathmin-like domain was 
92% and similarity was 99%. Stathmin is a coiled-coil phosphoprotein that regulates 
microtubule polymerization in a phosphorylation-dependent manner, and acts as a relay 
for intracellular signal transduction (Sobel, 1991; Belmont and Mitchison, 1996; 
Marklund et al, 1996). This region of the LYST gene may encode a coiled-coil protein 
interaction domain and may regulate microtubule-mediated lysosome trafficking. 
Intriguingly, a defect in microtubule dynamics has been documented previously in CHS 
(Oliver et al, 1975) and intact microtubules are required for maintenance of lysosomal 
morphology and trafficking (Matteoni and Kreis, 1987; Swanson et al, 1987; Swanson et 
al, 1992; Oka and Weigel, 1983). 

Mutation and expression analyses suggested that the largest Lyst splice variant 
(Lyst-I) represents the isoform of primary functional significance and that expression of 
the smaller isoform (Lyst-II) alone cannot compensate for loss of Lyst-I. This hypothesis 



91 

was supported by the genetic complementation experiments initiated in collaboration 
with Dr. Stephen Brandt. These studies revealed that some beige mouse fibroblasts, 
expressing full-length Lyst-I, demonstrated more peripheral distribution in the cytoplasm 
and slight reduction of the size of the stained granules. Such changes were not visible in 
cells expressing the smaller Lyst-U isoform. However, since the effects of the 
complementation were not very prominent and were not observed in all cells expressing 
Lyst-I, the significance of the described genetic complementation is questionable. 

Comparison of the relative abundance of LYST gene transcripts in human tissues 
at different developmental stages revealed an overlapping but distinct pattern of 
expression (Fig. 9). A quantitative estimate of the relative expression of the smaller 
mRNA isoforms was generated by comparing the relative hybridization intensity 
obtained with a probe specific for the large isoform with that obtained with a probe that 
hybridizes to all LYST transcripts. Large isoform transcripts predominated in thymus, 
fetal thymus, spleen and brain (with the exception of amygdala, occipital lobe, putamen 
and pituitary gland). Large and small mRNA transcripts were abundant in the latter brain 
tissues, peripheral blood leukocytes and bone marrow. However, in several tissues, only 
the small isoforms were expressed, e.g. in fetal heart, salivary gland, kidney, lung, and 
fetal brain. The developmental pattern of mRNA isoform expression in brain was 
particularly interesting, since only the small isoforms were expressed in fetal brain, 
whereas the largest isoform predominated in many regions of the adult brain. 



92 

Identification of Mutations in Patients with Chediak-Higashi Syndrome and in Beig e 

Mice 

Mutation analysis was performed in order to provide additional evidence that 
LYST, resp. Lyst is indeed the gene responsible for the CHS in human and for the beige 
mutation in mouse, as well as to determine whether a genotype-phenotype correlation 
could be established between different genetic defects and specific disease 
manifestations. Novel mutations were identified within the coding domain of LYST in 
several CHS patients (Table 4 and Fig.3). The genetic lesions in three patients (370, 372, 
373) were C-»T substitutions that resulted in premature termination. Another patient was 
heterozygous for a dinucleotide deletion that results in a frameshift and premature 
termination. Interestingly, all bg and LYST mutations identified to date are predicted to 
result in the production of either truncated or absent proteins, suggesting that missense 
mutations may not be likely to cause CHS. Unlike Fanconi anemia, type C (Yamashita et 
al., 1996) there does not appear to be a correlation between the length of the truncated 
proteins (which may or may not be stable) with clinical features or disease severity in 
CHS patients, given that a CHS patient with mild manifestations has a truncating 
mutation early in the protein. However, until the other mutant allele in the compound 
heterozygote patients is identified, and the exact effects of each mutation at the protein 
level are characterized, such correlation is imprecise. 

Mutation and expression analyses were also useful in addressing the important 
question about the biological relevance of the 2 main LYST transcripts in regulating 
lysosomal trafficking and in preventing Chediak-Higashi syndrome. The distribution of 



93 

expression of mRNA isoforms in human tissues was consistent with the pattern of clinical 
features observed in CHS patients: transcripts were most abundant in peripheral blood 
leukocytes, bone marrow, thymus, lymph node, spleen and brain, and common clinical 
features among CHS patients are immune deficiency, platelet storage pool deficiency and 
neurologic manifestations. Northern blots suggested that an ~ 13 kb mRNA 
(corresponding to the largest isoform) is severely reduced in abundance in two CHS 
patients (Fig. 3). A 4.4-kb band (corresponding to a smaller isoform), however, was 
present in normal abundance in mRNA from these patients. These results suggest that in 
some patients, Chediak-Higashi syndrome results from loss of the protein encoded by the 
largest isoform. Almost all of the mutations identified in CHS patients to date are within 
the region of the coding domain that is common to all isoforms (Barbosa et al, 1996; 
Nagle et al, 1996). However, in one CHS patient described by Karim et al, 1997, there 
was a frameshift (at codon 3197) leading to premature termination in a region of the 
molecule that belongs only to Lyst-I. In addition, the mouse bg 10 mutation results in the 
generation of a premature stop codon in Lyst-l that is unlikely to affect Lyst-ll mRNA 
processing (Perou et al, 1996b). Together, these results suggest that defects in the full- 
length mRNA alone can elicit Chediak-Higashi syndrome and that expression of the 
smaller isoform alone cannot compensate for loss of the largest isoform. Although we 
were not able to establish a more precise genotype-phenotype correlation between 
different genetic defects and specific disease manifestations, by identifying mutations in 
several CHS patients and bg alleles we did confirm that LYSTILyst is indeed the gene 
responsible for the Chediak-Higashi syndrome. 



94 

Identification and Characterization of Lyst2. a Brain-Specific Member of the Chediak- 

Higashi Syndrome Gene Family 

In an effort to identify genes similar to LYST, that may play roles in the regulation 
of vesicular trafficking, we have isolated a novel gene (Fig. 11) in human (LYST2) and 
mouse {Lyst2), that exhibits significant sequence identity, similar putative protein 
domains, and a comparable mRNA size (-13 kb) to the Chediak-Higashi syndrome 
cDNA (LYST). The segment of greatest sequence similarity between LYST and LYST2 
(>50% predicted amino acid identity) is located within the BEACH domain of LYST, a 
region exhibiting sequence conservation to ORFs from S. cerevisiae and C.elegans, and 
the human cell division control protein 4-related protein (Nagle et al, 1996). Although 
the C-terminal residues of LYST2 are not well conserved with LYST, they do have a 
similar predicted secondary structure. This region of LYST contains WD repeats and is 
predicted to assume a propeller secondary structure, similar to the B-subunit of 
heterotrimeric G proteins. Furthermore, the stop codons of mouse Lyst and human LYST2 
occur approximately the same distance from the matching region. There is also high 
degree of homology (96.2 % identity in 557 amino acids) between human LYST2 and 
mouse Lyst2 proteins. Based upon these observations, LYST2 is suggested to be a novel 
member of the LYST gene family. Since the 5' end of LYST2 has not been identified yet, 
the level of homology between LYST and LYST2 may change as new sequence is 
obtained. 

While Lyst maps on mouse Chr 13 (Kingsmore et al, 1996a; Kingsmore et al, 
1 996b), Lyst2 was mapped by linkage analysis between D3MU21 and D3MU22 on mouse 



95 

Chr 3 (Fig. 12). The only known mouse mutation located in this chromosomal region is 
blebs (my) at ~ cM 29.9. Blebs mice exhibit some abnormalities (e.g., pseudencephaly, 
midcerebral lesions, renal agenesis and hydronephrosis) that correspond to the organs 
with most abundant Lyst2 expression (brain and kidney). However, Lyst2 is not a very 
likely candidate for the my gene because blebs mice have numerous defects (split 
sternum, preaxial Polydactyly and syndactyly of the middle digits; eye, skin and hair 
abnormalities; ectopia viscerum) in organs, other than brain and kidney. In addition, my 
maps very close to the distal microsatellite marker (D3MU22 at ~ cM 33.7) used for the 
genetic mapping ofLyst2. 

In human, LYST was shown to map on Chr 1 (Fukai et al, 1996; Barrat et at, 
1996), while LYST2 was mapped in a somatic cell hybrid panel to human Chr 13 (Fig. 
13). Comparative analysis of the genome organization of human and mouse (Yui, 1997) 
did not reveal any conserved region between mouse chromosome 3 and human 
chromosome 13. Therefore, the determination of the precise localization of LYST2 on 
human chromosome 13 may potentially lead to the discovery of a new syntenic group 
between these autosomes. 

Northern blot analysis of human and mouse tissues (Fig. 14) suggested that the 
transcription of LYST2 had a similar complexity to that of LYST, with several, 
alternatively spliced isoforms. The size of the largest and most abundant LYST2 mRNA 
isoform is ~13 kb, which is very similar to the size of the largest LYST isoform. There is 
also evidence that smaller LYST2 transcripts (resembling in size those of LYST) exist, at 
least in mouse brain, but possibly in other mouse and human tissues. In contrast, there 
were considerable differences between Lyst and Lyst2 in their tissue distribution of 



96 

expression. While Lyst is expressed relatively uniformly in different normal mouse 
tissues (Barbosa et al, 1996), Lyst2 exhibited abundant expression only in brain. In 
addition, smaller Lyst transcripts (~4 kb) were much more abundant (Barbosa et al, 
1996) than those of Lyst2 (Fig 14a). 

The expression patterns of LYST and LYST2 in human tumor cell lines were also 
different (Fig. 14b). While the predominant LYST isoform is -3-4 kb, the only LYST2 
transcript is -13 kb. Similarly, in normal human immune tissues, the only visible LYST2 
isoform was -13 kb, while the most abundant LYST transcript was -3-4 kb. In both 
normal and tumor human tissues the expression of LYST was more uniform than that of 
LYST2. 

Analysis of the relative abundance of LYST and LYST2 transcripts in 50 normal 
human tissues demonstrated that there are considerable differences in the expression 
patterns (Fig. 15). LYST exhibited highest levels of expression in tissues of the immune 
system (e.g., adult and fetal thymus, peripheral blood leukocytes, bone marrow, spleen) 
and in some adult brain regions (e.g., cerebellum, medulla oblongata, frontal lobe, 
cerebral cortex, hippocampus). In contrast, LYST2 was expressed primarily in whole brain 
(adult and fetal), in most brain regions and in kidney, and demonstrated low relative 
expression in the immune system organs. 

Notably, there were some tissues, where LYST2 was expressed, but no LYST 
transcripts were detected, e.g., kidney, fetal brain, fetal heart, salivary gland and lung. 
However, there were also organs with high relative abundance of LYST mRN A and low 
relative expression of LYST2, mainly tissues of the immune system: thymus (adult and 



97 

fetal), peripheral blood leukocyte, bone marrow, spleen, lymph node, as well as in 
placenta and testis. 

It is unlikely that LYST2 probes cross-hybridize to LYST sequences because of the 
unambiguous genetic mapping and because of the observed expression differences 
between the two genes. 

In general, LYST demonstrated more uniform pattern of relative expression than 
LYST2, which was expressed in high levels only in brain and to a lesser extent in kidney. 

We identified a novel gene in human (LYST2) and mouse (Lyst2) that appears to 
be a relative of the Chediak-Higashi syndrome gene, based on sequence similarity, 
predicted protein structure and on similar transcript size. Comparison of the relative 
abundance of LYST2 and LYST mRNAs suggests that LYST2 is expressed abundantly 
only in brain and therefore it may represent a brain-specific member of the Chediak- 
Higashi syndrome gene family. These results suggest that these two genes may perform 
similar functions in different tissues: while LYST is expected to participate in the 
regulation of lysosomal trafficking and degranulation primarily in immune tissues, LYST2 
may play a similar role in cells of the nervous system. This hypothesis is supported by the 
yeast two-hybrid identification of a protein from the 14-3-3- family that interacts with 
both LYST and LYST2 (see below). 14-3-3 proteins have been shown to be involved in 
the regulation of Ca 2+ -dependent exocytosis of secretory vesicles (Chamberlain et al, 
1995; Roth et al, 1995), as well as in signal transduction (Fu et al, 1994; Reuther et al, 
1994). 



98 

Identification of Proteins that Interact with the CHS Protein. Using a Yeast Two-Hybrid 

Approach 

In a further effort to elucidate the function of the CHS protein, a modified yeast 
two-hybrid system was used for identification of proteins that interact with LYST and 
LYST2. Fourteen overlapping cDNA fragments, covering the entire coding domain of 
LYST and 4 fragments covering the known LYST2 sequence, were PCR amplified and 
cloned in binding- and activation-domain vectors containing different selectable markers. 
The resulting constructs were used as baits in forward and reverse screens of a human 
fetal brain cDNA library. Numerous tests (for bait self-activation, prey self-activation, 
non-specific binding, sequencing of all baits, matrix mating, P-galactosidase tests for 
haploids and diploids) were performed to determine the specificity of the detected 
baitprey interactions. 

A total of 24 interacting sequences (11 identical to published proteins, 2 highly 
homologous to published ESTs and 1 1 novel) passed all confirmation tests (Table 5). 
Interestingly, several of the 1 1 previously published proteins participate in the regulation 
of vesicular transport: 

1 . 14-3-3 proteins - a family of acidic proteins with molecular masses of- 30 kDa 
that are highly conserved over a wide range of eukaryotic organisms, including plants, 
yeast, insects, amphibians and mammals. Highest levels of 14-3-3 proteins were found in 
brain, but all animal and plant tissues examined to date contain several isoforms (Aitken 
et ah, 1992, 1995). Several important functions of 14-3-3 proteins have been described: 



99 

- Activation of tyrosine and tryptophane hydroxylases, the rate-limiting enzymes 
involved in neurotransmitter (catecholamine and serotonin) biosynthesis (Ishimura et al, 
1987). Although tyrosine hydroxylase was activated by PKC and c-AMP-dependent 
protein kinase phosphorylation, the 14-3-3 protein was necessary for activation after 
phosphorylation by Ca 2 7calmodulin-dependent (CAM) kinase II. 

- Regulation of protein kinase C (PKC) activity. There have been conflicting 
reports over the role of 14-3-3 as regulators of PKC. Toker et al, 1990, 1992 found that 
14-3-3 inhibited PKC, while Isobe et al, 1992 showed that 14-3-3 activated PKC 
approximately 2-fold. Nevertheless, interaction of 14-3-3 with PKC is established and it 
may be that the interaction of different 14-3-3 isoforms with PKC is responsible for the 
different results obtained. In addition, 14-3-3 proteins are phosphorylated by PKC itself, 
but not by a wide range of other protein kinases. 

- Interaction and activation of Raf protein kinase, a convergent point for different 
signals that regulate growth factor and hormone effects on differentiation and 
proliferation (Fu et al, 1994). Interactions of 14-3-3 with other signal transduction 
molecules have been described (Reuther et al, 1994; Burbelo and Hall, 1995; Morrison, 
1994), suggesting a role of the 14-3-3 family as modulators of signaling proteins. 

- Stimulation of Ca 2+ -dependent exocytosis in adrenal chromaffin cells (Burgoyne 
et al, 1993). This action of 14-3-3 is potentiated by PKC, which suggests a role for 14-3- 
3 in PKC-mediated control of exocytosis. These results are supported by the observation 
of membrane-bound 14-3-3 proteins, found at high concentration on the membrane of 
synaptic vesicles (Martin et al, 1994). In addition, 14-3-3 proteins have been shown to 
selectively stimulate the release of arachidonic acid from choline and ethanolamine 



100 

glycerophospholipids (Zupan et al, 1992). Arachidonic acid stimulates secretion from 
many cell types, which may be relevant to the role of 14-3-3 in exocytosis. In plants, 14- 
3-3-mediated increase in exocytosis has been proposed as a major mechanism involved in 
defense responses (Aitken et al, 1992). By using stage-specific assays for exocytosis in 
chromaffin cells, it has been shown that 14-3-3 proteins, similarly to oc-SNAP, enhanced 
exocytosis by acting in an early, priming stage (Chamberlain et al, 1995). However, the 
effects of 14-3-3 and oc-SNAP showed different Ca 2+ -dependencies and time courses, 
suggesting that they had distinct actions in priming. The time courses of action of 14-3-3 
and a-SNAP would be consistent with 14-3-3 proteins activating a process which must 
occur only once for increased secretion, whereas a-SNAP may be needed continuously 
during secretion to facilitate multiple rounds of vesicle recruitment, docking and fusion. 
It has been demonstrated that the stimulation of catecholamine release by 14-3-3 proteins 
was abolished by prior incubation with the actin filament stabilizing drug phalloidin 
(Roth and Burgoyne, 1995). The cortical actin network was disassembled and actin was 
reorganized into intracellular foci following treatment with 14-3-3 proteins. These data 
suggest that 14-3-3 proteins enhance catecholamine release by reorganization of the 
cortical actin barrier to allow increased availability of secretory vesicles for exocytosis. It 
has been shown that other stimuli that activate exocytosis lead to actin reorganization in 
other cell types, e.g. in mast cells (Koffer et al, 1990) and parotid acinar cells (Perrin et 
al, 1992). Roth et al, 1994, reported that 14-3-3 is associated with granule membranes 
and that it can bind phospholipid vesicles and induce their aggregation. Since membrane 
fusion in exocytosis involves proteins able to modify or reorganize lipids within the 



101 

plasma and granule membranes, the ability of 14-3-3 to bind phospholipids and to 
aggregate phospholipid vesicles suggests that 14-3-3 proteins could play a role in 
membrane events during exocytosis. 

The functions of 14-3-3 proteins in vesicular transport are in close agreement with 
the expected roles of the CHS protein. As described in the INTRODUCTION of this 
dissertation, there are numerous examples of impaired vesicle trafficking in CHS - 
melanosomes are affected in melanocytes, dense granules in platelets, lysosomes and 
secretory vesicles in various immune cells. These defects indicate that LYST may 
participate in the regulation of intracellular vesicular transport and in exocytosis, where 
14-3-3 proteins have been shown to perform important functions. These observations 
support the detected association between the CHS protein and 14-3-3. Similarly to the 
modulation of protein kinase C by 14-3-3, a rapid down-regulation of membrane-bound 
PKC activity following short-term activation has been described in beige macrophages, 
NK cells and polymorphonuclear leukocytes (Ito et ah, 1988, 1989). In addition, a 
concavalin A (Con A)-induced, PKC-dependent cap formation was significantly 
increased in beige PMNs compared to that in control cells (Sato et ah, 1990). The decline 
in PKC activity and the abnormally enhanced cap formation were both abolished by 
pretreatment of cells with leupeptin, an inhibitor of calpain (a Ca 2+ -dependent thiol 
proteinase that hydrolyses PKC). Furthermore, the treatment with leupeptin improved the 
impaired NK cell activity from bglbg mice, whereas the NK cells from bg/+ and +/+ 
were not affected. These findings suggest that in CHS there is a dysregulation of protein 
kinase C activity that may alter other signal transduction pathways and cause functional 
cellular defects. The involvement of 14-3-3 and the CHS protein in both vesicular 



102 

transport and signal transduction indicate that these two molecules function in common 
pathways and supports the LYST: 14-3-3 interaction detected by the yeast two-hybrid 
system. 

2. Casein kinase II (CK2, CKII) - serine/threonine protein kinase that is present in 
the nucleus and cytoplasm of all eukaryotic cells that have been studied. CK2 is a 
heterotetramer with a molecular mass of- 130 kDa, composed of 2 types of subunits (a 
and P) and has the general structure a 2 P 2 or aa'P 2 . The a and a' are catalytically active, 
while the P subunit is inactive by itself but it stimulates the catalytic activity of a 5- to 
1 0-fold, causes stabilization of a and more importantly, can change the interactions with 
substrates and inhibitors (Allende and Allende, 1995). Casein kinase II is unusual among 
the protein kinases in that it can use both ATP and GTP as phosphate donors. There are 
more than 100 different protein substrates that are phosphorylated by CK2, many of them 
involved in DNA replication, transcription and signal transduction. It has been 
demonstrated that one of the major brain substrates for CK2 is synaptotagmin, an 
abundant synaptic vesicle protein that has been implicated in vesicle docking and fusion 
with the plasma membrane (Davletov et al., 1993). Synaptotagmin binds Ca 2+ and 
phospholipids at concentrations that are thought to occur physiologically in the nerve 
terminal during depolarization, suggesting that it may be a regulatory Ca 2+ -binding 
protein involved in synaptic vesicle exocytosis. Synaptotagmin is part of a tetramer with 
multiple Ca 2+ -binding sites and represents the only known Ca 2+ -binding protein on the 
synaptic vesicle surface and the first membrane-bound putative regulatory Ca 2+ -binding 
protein (Davletov et al, 1993). Ca 2+ causes an increase in the phosphorylation of 



103 

synaptotagmin by CK2 and not by a Ca 2+ -dependent kinase. The mechanism of this 
activation is unclear, but it could be related to the fact that synaptotagmin is a Ca 2+ - and 
phospholipid-binding protein that may undergo Ca 2+ -dependent conformational changes. 
Synaptotagmin has been reported to interact with several different plasma membrane 
proteins, including syntaxin, and may have a role in the docking of synaptic vesicles. It is 
the only one of the major synaptic vesicle proteins with a putative function in vesicular 
trafficking that is also found on other secretory vesicles with Ca 2+ -regulated exocytosis 
(Perin et al, 1991). This suggests that if synaptotagmin functions in the final steps of 
vesicle exocytosis, these steps may be similar in different secretory pathways. The 
potential significance of the phosphorylation of synaptotagmin by casein kinase II is 
supported by the finding that synaptotagmin is a very efficient substrate (better than 
casein) for CK2 and not for other kinases (Davletov et al, 1993). The phosphorylation 
site is present in both synaptotagmins I and II and is evolutionarily conserved in different 
species. Bennet et al, 1993, demonstrated that casein kinase II not only phosphorylates 
synaptotagmin, but also interacts with a domain of synaptotagmin that is distinct from the 
phosphorylation site. The functional significance of the CK2: synaptotagmin association 
is unclear but it probably does not simply represent an enzyme: substrate interaction and 
may have a modulatory role in nerve terminal function. 

The involvement of casein kinase II in synaptic vesicle trafficking was further 
supported by the findings of Bennet et al, 1993, and Hiding and Scheller, 1996, that 
CK2 phosphorylates syntaxin and possibly synaptobrevin, the two main neuronal 
SNAREs, called also t-SNARE and v-SNARE, respectively. The interaction between 
syntaxin and synaptobrevin is considered one of the critical steps in vesicle docking and 



104 

fusion. In general, protein phosphorylation has been well established as a regulatory 
mechanism for vesicle transport: the synaptic vesicle protein synapsin by 
Ca 2 7calmodulin-dependent protein kinase II regulates its interaction with both synaptic 
vesicles and actin filaments (Schiebler et ah, 1986; Benfenati et ah, 1992). This in turn 
may regulate the availability of synaptic vesicles for release. Similarly, phosphorylation 
of n-secl by protein kinase C has been shown to regulate the n-secl:syntaxin binding 
(Fujita et al., 1996). CK2 is also indirectly involved in the regulation of synaptic vesicle 
transport by phosphorylating calmodulin and PKC, which are known to play important 
roles in this process. Casein kinase II could be copurified with clathrin-coated vesicles 
and it could phosphorylate the P-light chain of clathrin, implicating CK2 in vesicular 
trafficking in general. 

LYST has several sites of potential phosphorylation by casein kinase II. However, 
the CK2:LYST interaction detected by the yeast two-hybrid system is not one of enzyme 
and substrate since the p-subunit of CK2, which interacts with LYST, has a regulatory 
and not a catalytic activity. This does not, however, exclude the possibility that LYST is 
phosphorylated by the catalytic a-subunit of CK2. A similar example of distinct 
phosphorylation and interaction between synaptotagmin and casein kinase II was 
described above. The involvement of CK2 in the regulation of synaptic vesicle exocytosis 
and vesicular trafficking, where LYST is expected to perform its function, further 
supports the detected interaction between these 2 molecules by the yeast two-hybrid 
method. 



105 

3. Calmodulin (CaM) - an abundant Ca 2+ -binding protein found in all eukaryotic 
cells that have been studied. It is a highly conserved, single polypeptide chain with four 
high-affinity Ca 2+ binding sites, and it undergoes a conformational change when it binds 
Ca 2+ . CaM functions as a multipurpose intracellular Ca 2+ receptor, mediating many Ca 2+ - 
regulated processes. Calmodulin has no enzyme activity by itself but acts by binding to 
other proteins and altering their activity. Among the targets regulated by Ca 2 7calmodulin 
are various enzymes and membrane transport proteins. In many cells, for example, 
Ca 2 7calmodulin binds to and activates the plasma membrane Ca 2+ -ATPase that pumps 
Ca 2+ out of the cell. Most effects of Ca 2 7calmodulin, however, are more indirect and are 
mediated by Ca 2 7calmodulin-dependent protein kinases (CaM-kinases) (Alberts et al., 
1994). These enzymes phosphorylate serines or threonines in proteins and the response of 
a target cell to an increase in free Ca 2+ concentration depends on which CaM-kinase- 
regulated target proteins are present in the cell. The best studied example is CaM-kinase 
II, which is found in all animal cells but is especially enriched in the nervous system 
where it is highly concentrated in synapses. When neurons that use catecholamines as 
their neurotransmitters are activated, the influx of Ca 2+ through the plasma membrane 
stimulates the cell to secrete their neurotransmitter. The Ca 2+ influx also activates CaM- 
kinase II to phosphorylate, and thereby activate, tyrosine hydroxylases, the same enzymes 
that are activated by the 14-3-3 proteins, as described above. CaM-kinase II can function 
as a molecular memory device, switching to an active state when exposed to 
Ca 2 7calmodulin and then remaining active, due to autophosphorylation, even after Ca 2+ is 
withdrawn. It has been shown that CaM-kinase II phosphorylates a number of nerve 



106 

terminal proteins, including syntaxin, synaptobrevin, SNAP-25, aSNAP, NSF and 
synaptotagmin, indicating a significant involvement of this kinase in neurotransmitter 
release (Hirling and Scheller, 1996). 

There is convincing evidence that calmodulin is involved in neurotransmitter 
release and secretion in general. It is present in nerve terminals and is associated with 
synaptic vesicles (DeLorenzo, 1981), as well as with other secretory vesicles: platelet oc- 
granules, chromaffin vesicles and posterior pituitary secretory granules. With the use of 
different antagonists, calmodulin has been implicated in secretion in a number of cell 
systems, including adrenal medulla, endocrine pancreas, anterior pituitary, platelets, mast 
cells, adrenal cortex, nerve terminals, and leukocytes (Trifaro et ah, 1992). Calmodulin is 
one of the proteins from a crude cytosol fraction that will bind to chromaffin granules in 
the presence of Ca 2+ . This binding involves specific proteins on the cytoplasmic surface 
of the granules. Two major Ca 2+ -dependent calmodulin binding proteins of the 
chromaffin granule membrane have been described and one of them has been identified 
as the integral membrane protein synaptotagmin (p65), which also interacts with and is 
phosphorylated by casein kinase II, as described above. Although the significance of the 
granule membrane-CaM binding is unclear, the fact that synaptotagmin is a component of 
synaptic and secretory vesicles from several types suggests that calmodulin binding to 
this protein may participate in the control of exocytosis. In agreement with these results, 
high-affinity binding sites for calmodulin have been found in coated vesicles (Moskowitz 
et ah, 1982). When anti-calmodulin antibodies are introduced into chromaffin cells, 
secretion is inhibited, indicating involvement of CaM in exocytosis. Similar results have 



107 

been obtained with anti-calmodulin antibodies in other systems as well (Trifaro et al., 
1992), supporting a role for CaM in secretion. Fusion experiments carried out with 
plasma membranes and anterior pituitary secretory granules or pancreatic granules have 
indicated the requirement of calmodulin for vesicle-plasma membrane fusion processes 
(Watkins and Cooperstein, 1983). Addition of calmodulin to permeabilized chromaffin 
cells increased Ca 2+ -dependent norepinephrine release in a dose-dependent and CaM- 
specific manner. The enhanced norepinephrine release was inhibited by tetanus toxin, 
indicating that calmodulin plays an important role in exocytosis from chromaffin cells 
(Matsuda et al., 1994). Similarly, exocytosis of cortical granules caused by an increase of 
Ca 2+ content immediately upon fertilization of sea urchin eggs appears to be controlled by 
Ca 2 7calmodulin. Incubation with antibodies against CaM causes a loss of sensitivity to 
Ca 2+ . Sensitivity is restored when calmodulin is added in excess to inhibited preparations. 
Cortical exocytosis is also inhibited by CaM-antagonists added to sea urchin eggs 
(Kreimer and Khotimchenko, 1995). By using stage-specific assays for ATP-dependent 
priming and Ca 2+ -mediated triggering of exocytosis, Chamberlain et al., 1995, 
demonstrated that calmodulin acted in the ATP-independent triggering step and 
stimulated exocytosis. An indirect participation of CaM in neurotransmitter release may 
involve calcineurin, a Ca 2 7calmodulin-dependent protein phosphatase that is enriched in 
neural tissues. Among the serine/threonine protein phosphatases, calcineurin is unique in 
its requirement for Ca 2 7calmodulin activation. A variety of ion channels (both ligand- 
and voltage-gated) that play major roles in regulating neuronal excitability and 
transmitter release appear to be negatively regulated by calcineurin. In addition, the 



108 

release of glutamate from presynaptic terminals and hormone secretion in pancreatic (5 
cells and pituitary cells also seem to be under negative regulation by calcineurin (Yakel, 
1997). A similar indirect role of calmodulin in exocytosis was shown by Burgoyne et ah, 
1986, through modulation of the activity of caldesmon, a CaM-regulated actin-binding 
protein. It is likely that increased cytosolic Ca 2+ following stimulation results in solation 
of actin filaments crosslinked by caldesmon, and the subsequent binding of caldesmon to 
the chromaffin granule regulates interactions of the vesicles with the plasma membrane or 
the cytoskeleton and leads to exocytosis. 

The direct and indirect involvement of calmodulin in synaptic vesicle exocytosis 
and in secretion in general is in close agreement with the expected roles of LYST. 
Furthermore, the findings that CaM is phosphorylated by casein kinase II (another Lyst- 
interacting protein) and that through CaM-kinase II it regulates the function of tyrosine 
hydroxylases (also activated by the LYST-interacting 14-3-3 proteins) and of several 
nerve terminal proteins (syntaxin, synaptotagmin, synaptobrevin, SNAP-25, etc.), further 
supports the detected LYSTxalmodulin interaction and unites all these LYST-interacting 
proteins in a common network involved in vesicular trafficking regulation. 

4. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) - a 1 1 5-kDa 
protein that was originally discovered due to its rapid phosphorylation in cells treated 
with hepatocyte growth factor (Komada and Kitamura, 1994). Hrs contains a conserved 
double zinc finger-like motif, as well as a proline-rich, proline- and glutamine-rich, and 
coiled-coil regions that may mediate protein-protein interactions. It is expressed in a 
variety of adult mouse tissues and at different stages of mouse whole embryos (Komada 



109 

and Kitamura, 1995). Tyrosine phosphorylation of Hrs was also induced in cells treated 
with epidermal growth factor and platelet-derived growth factor suggesting that Hrs may 
play a role in the signal transduction pathways of different growth factors in a variety of 
cell types. 

Bean et al, 1997, demonstrated that Hrs is an ATPase and that it interacts with 
SNAP-25 (synaptosomal-associated protein 25), a nerve terminal protein that resides on 
the internal surface of the plasma membrane and interacts with syntaxin (t-SNARE), 
synaptobrevin (v- SNARE) and a SNAP to form a core complex thought to be an 
intermediate in a biochemical pathway that is essential for vesicular transport. The 
binding of Hrs to SNAP-25 is inhibited by Ca 2+ in the physiological concentration range 
that supports synaptic transmission. Hrs is expressed abundantly throughout the brain and 
is present at nerve terminals. Recombinant Hrs inhibits Ca 2+ -triggered noradrenaline 
release from permeabilized cells in a dose-dependent and saturable manner. The Hrs- 
SNAP-25 binding was inhibited by Zn 2+ , and the previously reported enhancement of 
neurotransmitter release induced by Zn 2+ suggests that the inhibition of Hrs-SNAP-25 
binding may facilitate transmitter release. These data provide a functional demonstration 
of the ability of Hrs to regulate the secretory processes through Ca 2+ - and nucleotide- 
dependent modulation of vesicle trafficking protein complexes. It is possible that Hrs 
plays a regulatory role in vesicle docking through its ATPase activity, and in later stages 
of secretion, possibly membrane fusion, as a result of calcium-regulated interaction with 
SNAP-25. This hypothesis was supported by the finding that Hrs is peripherally 
associated with the cytoplasmic surface of early endosomes (Komada et al, 1997). By 



110 

subcellular fractionation, Hrs was recovered both in the cytoplasmic and membrane 
fractions. These results, together with the finding that Hrs is homologous to Vps27p, a 
molecule essential for protein trafficking through a prevacuolar compartment in yeast, 
suggest that Hrs is involved in endosomal transport. It is hypothesized that the growth 
factor-bound receptor tyrosine kinase is internalized and delivered to the early endosome, 
where it phosphorylates Hrs residing on the cytoplasmic surface of the early endosome. 
Tyrosine phosphorylation activates Hrs and it stimulates the vesicle transport of the 
growth factor-receptor complex to the late endosome and lysosome. Therefore, Hrs may 
be a regulator of several types of vesicular functions (endo- and exocytosis) in a variety 
of cells. 

In addition to its role in the hepatocyte growth factor intracellular signaling 
pathway, Hrs was found to interact with STAM (signal transducing adaptor molecule), 
which was shown to be involved in cytokine-mediated intracellular signal transduction 
(Asao et ah, 1997). Tyrosine phosphorylation of Hrs was induced rapidly after 
stimulation with IL-2 and GM-CSF, as well as HGF. The mutual association sites of Hrs 
and STAM include highly conserved coiled-coil sequences, suggesting that their 
association is mediated by the coiled-coil structures. Exogenous introduction of Hrs 
significantly suppressed DNA synthesis upon stimulation with IL-2 and GM-CSF, while 
an Hrs mutant deleted of the STAM-binding site lost such suppressive ability, i.e. Hrs- 
STAM association is a prerequisite for such activity. These results suggest that Hrs 
counteracts the STAM function which is essential for cell growth signaling mediated by 
the cytokines. 



Ill 

The involvement of Hrs in both signal transduction and vesicular trafficking 
correlates well not only with the predicted function of LYST but also with the roles of the 
other LYST-interacting proteins detected by the yeast two-hybrid method. IL-2, which 
induces rapid tyrosine phosphorylation of Hrs, also activates the Shc/Grb2/Sos/Ras/ 
Rafl/mitogen-activated protein kinase cascade (Asao et al., 1997). Rafl has been shown 
to interact with 14-3-3 proteins (another Lyst-interacting protein), as described above. IL- 
2 also activates Fyn tyrosine kinase, and the SH2 domain of Fyn has been reported to 
interact with Rafl (Morrison, 1995), providing an additional link to 14-3-3 and, therefore, 
to LYST. This association is supported further by the finding of interaction between 14- 
3-3 and Bcr, a protein kinase that is involved in the same growth factor signaling 
Shc/Grb2/Sos/Ras/Rafl /MAP -kinase cascade. On the other hand, it has been 
demonstrated that Hrs is homologous to Vps27p, a yeast protein localized to the 
prevacuolar compartment considered to be the counterpart of mammalian endosomes. 
VPS27 is one of the genes whose disruption in S. cerevisiae dysregulates the traffic of 
endocytosed proteins and newly synthesized vacuolar proteins to vacuoles (a counterpart 
of mammalian lysosomes) and causes the accumulation of these proteins in a prevacuolar 
compartment. The finding that Hrs is localized to the cytoplasmic surface of early 
endosomes indicates that in mammalian cells Hrs may play a role similar to that of 
Vps27p in yeast. The involvement of Hrs in Ca 2+ -regulated secretion and its interaction 
with SNAP-25, a protein that functions in exocytosis of neurotransmitter vesicles, further 
supports the LYST:Hrs interaction detected by the yeast two-hybrid system. 

The rest of the previously published proteins, identified as LYST-interactants by 
the yeast two-hybrid screens, are not as obviously involved in vesicular trafficking and its 



112 

regulation, as the above described 14-3-3 proteins, casein kinase II, calmodulin and Hrs. 
However, for most of these proteins there are some functional and/or morphological 
indications that they may participate in the same pathways as LYST, 14-3-3, CK2, CaM 
and Hrs. 

1. Atrophin 1 - the gene responsible for dentatorubral pallidoluysian atrophy 
(DRPLA, Smith's disease), a rare autosomal dominant neurodegenerative disorder 
characterized by cerebellar ataxia, myoclonic epilepsy, choreoathetosis and dementia. 
The molecular defect in DRPLA is an expansion of a CAG trinucleotide repeat (encoding 
glutamine) in atrophin 1. The gene is expressed in many mammalian (but not in avian) 
species, in all brain regions tested, most abundantly in cerebellum. There is no detectable 
difference in the level or distribution of atrophin 1 expression between normal and 
DRPLA-brain, indicating that disease pathology may result from altered protein function 
(Margolis et al., 1996). Immunohistochemical studies demonstrated predominantly 
cytoplasmic signal with prominent granular and punctate staining within cell bodies and 
dendrites, suggesting that atrophin 1 may play a role in vesicle transport (Knight et al., 
1997; Yazawa et al, 1995). This hypothesis is supported by the finding of 2 atrophin- 
interacting proteins. Both have WW domains that may interact with PY motifs in 
atrophin 1. Expression of one of these proteins appears to be restricted to the brain, while 
the other is expressed in all tissues examined. One of the atrophin-interacting proteins is 
homologous to members of a family of E3 ubiquitin-protein ligases, including the yeast 
Rsp5 protein. Interestingly, rsp5 mutants are deficient in endocytosis (Ross et al., 1997), 
suggesting a possible involvement of atrophin 1 in vesicular transport and supporting the 
detected LYST:atrophin 1 interaction. 



113 

2. Imogen 38 - a novel -38 kDa protein recognized by a T cell clone from a type 
I diabetic patient. The protein is expressed at variable levels in many tissues and cell 
lines. There is some discrepancy regarding the subcellular localization of imogen 38 - 
Arden et al., (1996) showed that the mitochondrion is the major site of the protein 
(although there was some overlap of the antigen distribution with that of secretory 
granules), while Roep et al., (1990) determined that imogen 38 was associated with 
insulin secretory granules. Although the precise function of the protein is not known, its 
vesicular localization suggests a possible involvement in secretion, indirectly supporting 
the detected LYST: imogen 38 interaction by the yeast two-hybrid system. 

3. Embryonal Fyn substrate 2 (Efs2) - an isoform of the Efs gene, lacking the 
SH3 domain. Efs was originally discovered through its Fyn-binding capacity. Embryonal 
Fyn substrate has characteristic regions important in intracellular signal transduction - an 
SH3 domain, a cluster of putative ligands for SH2 domains and proline-rich sequences 
with SH3-binding consensus. The protein was named "embryonal Fyn substrate" because 
its RNA was most abundantly expressed in embryo, although expression was shown in 
many adult tissues as well (Ishino et al, 1997). The SH3 domain has been shown to 
mediate specific binding of Sosl to Grb2 and this important protein-protein interaction 
links the tyrosine kinase pathway initiated at the membrane surface to the Ras pathway. 
Efs2, which lacks the SH3 domain, may participate in the regulation of coupling of these 
signaling proteins. The associations of Efs/Efs2 with the tyrosine kinase Fyn and with the 
Grb2/Sosl/Ras pathway indicate its possible involvement in the same processes as Hrs 
and 14-3-3, two other LYST-interacting proteins. 



114 

4. Importin P-subunit - a 90-kDa protein that binds the 60-kDa a-subunit, 
forming a heterodimer, called importin, or karyopherin. The a-subunit recognizes the 
nuclear localization signal (NLS) of a cargo protein (called karyophile) that is to be 
transported from the cytoplasm through a nuclear pore into the nucleus, while the P- 
subunit first docks the a/p/karyophile complex at the cytoplasmic face of the nuclear pore 
and then mediates the translocation by interacting with the pore proteins. The small 
GTPase Ran, as well as a cofactor called plO, pi 5 or NTF-2, are required for nuclear 
import. RanGTP dissociates a from P, and may promote the translocation of the 
a/p/karyophile complex across the pore and the release of the complex from the nuclear 
side of the pore (Laskey et ai, 1996). Interestingly, both importin a and P consist largely 
of repeated domains, the so called arm repeats, which are also present in LYST. These 
repeats are found in several functionally unrelated proteins, including the founder 
member of the family, the Drosophila armadillo protein (Peifer et ai, 1994). Importin a 
has 8 arm repeats, while importin p has 1 1 more divergent repeats, although the precise 
number depends on how the domains are defined. The NLS binds directly to the arm 
repeat region of the a-subunit. Besides the presence of the arm repeats, the only other 
common feature of LYST and importin p is that both proteins are involved in protein 
transport, although importin plays a role in nuclear transport, while LYST is expected to 
participate in vesicular protein trafficking. 

5. Estrogen receptor-related protein (ERR1) - a member of the nuclear hormone 
receptor superfamily of zinc finger transcription factors. The ERR1 differs from the 
classical nuclear hormone receptors in that it has no known ligand, making it an "orphan 



115 

receptor". It has been shown that the transcription activity mediated by the glucocorticoid 
receptor was strongly suppressed by estrogen receptor-related protein in a cell- and 
receptor-specific manner (Trapp and Holsboer, 1996). However, ERR1 activates estrogen 
receptor-mediated response of the lactoferrin gene promoter by binding to a regulatory 
DNA sequence upstream of the estrogen response element (Yang et al, 1996). It has also 
been demonstrated that ERR1 interacts with the estrogen receptor through a direct protein- 
protein contact, suggesting that ERR1 may participate in estrogen stimulation of the 
lactoferrin gene (and possibly in other estrogen-mediated pathways) and therefore ERR1 
may play a role in modulating immune responses and cell growth. 

6. DiGeorge syndrome-I (DGS-I) - one of the genes from the critical region of 
DiGeorge syndrome and velo-cardio-facial syndrome on human Chr 22qll. Both 
conditions could be caused by microdeletions in this minimal region. It has not been 
demonstrated that DGS-I is responsible for any of the features of DiGeorge syndrome 
(absence or hypoplasia of the thymus and parathyroid glands, cardiovascular anomalies 
and mild craniofacial dysmorphia) or velo-cardio-facial syndrome (cleft palate, cardiac 
anomalies, typical facies and learning disabilities). DGS-I does not show significant 
homology to any genes with known function and its role is unclear. There are 2 DGS-I 
isoforms: the small (~ 1.7 kb) is expressed in all tissues tested, while the large (~ 5.2 kb) 
is expressed in heart and skeletal muscle only (Gong et al, 1996). Since the available 
information about DGS-I is so limited, it is hard to discuss the interaction between LYST 
and DGS-I detected by the yeast two-hybrid method. 

The rest of the LYST-interacting proteins (total of 13) were novel (some were 
homologous to ESTs) and they were briefly discussed in the RESULTS chapter of this 



116 

dissertation. Notably, one of the interactants (IP-1 1) is a novel protein with homology to 
the chaperone TCP-1. Molecular chaperones guide the folding of newly synthesized 
proteins through a "molten globule" state to the final correct protein conformation. 
Eukaryotic cells have chaperone proteins of the hsp60 and hsp70 (heat-shock proteins) 
families. Different family members have specific subcellular distribution and function in 
different organelles, e.g., mitochondria contain their own hsp60 and hsp70 molecules that 
are distinct from those that function in the cytosol. A special hsp70 chaperone, called 
binding protein (BiP), not only helps in protein folding in the endoplasmic reticulum, but 
also keeps the resident proteins in the ER and thus out of the Golgi apparatus and the rest 
of the secretory pathway. In CHS cells there is compartmental miss-sorting of proteins 
such as elastase, P-glucuronidase, and cathepsin G (Holcombe et al, 1994). In addition, 
the low tyrosinase activity in giant melanosomes results from mislocalization and 
eventual extracellular secretion of tyrosinase. These observations support the detected 
LYST:IP-1 1 interaction since it is possible that the protein miss-sorting, demonstrated in 
CHS cells, is related to deficient TCP-1 -like chaperone function in the absence of LYST. 
Using the yeast two-hybrid system, LYST 2 was found to interact with 2 proteins 
- 14-3-3 and human brain factor 2. The finding of a protein (14-3-3) that interacts with 
both LYST and LYST2 supports not only the validity of the detected interactions by the 
yeast two-hybrid system, but also the predicted relationship between LYST and LYST2. 
Since the involvement of 14-3-3 in vesicular trafficking and signal transduction was 
already described above, only the role of HBF-G2 will be discussed here. 



117 

Human brain factor 2 (HBF-G2) - a member of the fork head family of 
transcription factors. It is expressed primarily in embryonic brain but also at lower levels 
in several adult tissues. HBF-G2 shows high homology to human brain factor 1 and to the 
retroviral oncogene qin (Wiese et ai, 1995). The cellular homologs of retroviral 
oncogenes represent important growth-regulatory genes. Although its function is not 
known, it is hypothesized that HBF-G2 plays a role in cell proliferation during embryonic 
brain development and possibly in some adult tissues. The findings that both LYST2 and 
HBF-G2 are expressed most abundantly in brain indirectly supports their interaction 
detected by the yeast two-hybrid system. 

All of the described IPs were identified by screening only a human brain cDNA 
library. Other LYST- and LYST2-interacting proteins could by isolated if other libraries 
(e.g., lymphoid) are screened. 

An attempt was made to group the previously published LYST- and LYST2- 
interacting proteins based on their known or predicted function (Figure 16). 

Ten of the 1 1 proteins could be categorized in only 2 overlapping sets: embryonal 
Fyn substrate 2, estrogen receptor-related protein and human brain factor 2 play roles in 
signal transduction; atrophin 1, imogen 38 and importin function in protein transport; and 
14-3-3 proteins, casein kinase II, calmodulin and Hrs are involved in both signal 
transduction and protein transport. The only LYST-interacting protein with currently 
unknown function is DGS-I but in the future, following its characterization, it may also 
be categorized in one of the above 2 groups. 



118 



PROTEIN TRANSPORT 



SIGNAL TRANSDUCTION 



ATROPHIN-1 

IMPORTIN-p 

IMOGEN 38(?) 




UNKNOWN FUNCTION 



Figure 16. A classification of the previously known LYST- and LYST2- 
interacting proteins based on their established or predicted function. Almost all of the IPs 
could be categorized in only 2 overlapping groups - protein transport and signal 
transduction. Four proteins (14-3-3, casein kinase II, calmodulin and Hrs) belong to both 
categories and provide a link between the other interacting proteins. 



119 

Figure 1 7 represents a simplified diagram of the interactions between L YST, 
LYST2 and 4 of their interacting proteins in the context of neurotransmitter vesicle 
docking/fusion with the presynaptic plasma membrane of a neuron. These 4 LYST-IPs 
(14-3-3, CK2, calmodulin and Hrs) were selected and included in this diagram because 
their roles in both signal transduction and vesicular transport are well established and 
these 4 proteins provide a link between the other LYST-IPs, categorized in either signal 
transduction or protein transport only. It is shown that LYST, 14-3-3, CK2, calmodulin, 
Hrs and other proteins known to be involved in vesicular trafficking and signal 
transduction form a common network of interacting molecules involved in the regulation 
of these processes. 

An independent way of confirming the detected LYST:IP interactions would be to 
perform coimmunoprecipitation using specific antibodies. While antibodies against some 
of the LYST-IPs are available, a high-quality LYST-specific antibody has proved 
difficult to raise, after several attempts by different groups. An antibody against the C- 
terminus of Lyst, generated in collaboration with Dr. Stephen Brandt, did recognize a 
protein with the expected size for Lyst in normal mouse cells. However, this antibody 
recognized a protein with the same size in bg/bg cells which are not supposed to produce 
full-length Lyst because of a LINE1 element retrotransposition leading to a premature 
termination. Therefore, it appears that this antibody is not Lyst-specific and cannot be 
used for coimmunoprecipitation experiments. Generation of an anti-LYST antibody and 
confirmation of the LYST:LYST-IP interactions would be an important step in the future 
characterization of the Chediak-Higashi syndrome gene and its products. 



120 



CYTOPLASM 



VESICLE 




OUT 



Figure 17. A simplified schematic representation of the interactions between 
LYST, LYST2, some of their interacting proteins and other relevant molecules in the 
context of neurotransmitter vesicle docking/fusion with the presynaptic plasma 
membrane of a neuron. All proteins form a common network of interacting molecules 
involved in the regulation of vesicle exocytosis. 



121 

In summary, it was demonstrated that each of the previously reported bg gene 
sequences are derived from a single gene with alternatively spliced mRNAs. Alternative 
splicing results in bg gene isoforms (Lyst-\ and Lyst-ll) that contain different 3' regions. 
Similarly to mouse, the human mRNA isoforms arise from incomplete splicing and 
retention of a transcribed intron that encodes the C-terminus of the predicted LYST 
protein. Additional splicing complexity of smaller isoforms exists - Lyst-lll isoform lacks 
exons a and p, while in Lyst-W, exons a, P and y are absent. Novel mutations were 
identified within the coding domain of LYST in several CHS patients and bg alleles. 
Interestingly, all bg and LYST mutations identified to date are predicted to result in the 
production of either truncated or absent proteins. Mutation and expression analyses 
suggested that defects in the full-length mRNA alone can elicit CHS and that expression 
of the smaller isoform alone cannot compensate for loss of the largest isoform. We have 
also identified a novel gene in human (LYST2) and mouse (Lyst2) that appears to be a 
relative of the CHS gene, based on sequence similarity, predicted protein structure and on 
similar transcript size. Comparison of the relative abundance of LYST2 and LKST mRNAs 
suggests that LYST2 is expressed abundantly only in brain and therefore it may represent 
a brain-specific member of the CHS gene family. Using a modified yeast two-hybrid 
system, a human cDNA library was screened with baits from the coding domains of 
LYST and LYST2. Several proteins, which play important roles in protein transport and 
signal transduction, such as 14-3-3, CK2, calmodulin and Hrs, were found to interact with 
LYST and/or LYST2. Many of the interacting proteins could be linked in a common 
pathway that regulates vesicular trafficking and degranulation. 



122 

The molecular characterization of the Chediak-Higashi syndrome gene is of 
medical significance for several reasons. It will lead to improved understanding of CHS 
pathology and may suggest treatments that are not currently being considered. Targets for 
drugs that will limit degranulation and thereby ameliorate organ damage in inflammatory 
diseases may be developed as a result of characterization of molecules that regulate 
lysosomal secretion from granular cells. The observation that bg mice are protected from 
immune complex nephritis in the setting of systemic autoimmune disease (Clark et al, 
1982) suggests that selective blockade of Lyst may be a novel mode of protection from 
immunopathology in inflammatory diseases such as lupus nephritis. Finally, Lyst 
characterization may contribute to the molecular dissection of the associated cancer 
predisposition and clarify the role of natural immunity in surveillance against tumor 
development and metastasis. 



123 



APPENDIX 
SEQUENCES OF MOLECULES INTERACTING WITH LYST AND LYST2 

For all previously known proteins, GenBank accession numbers are shown in 
parentheses. The arrows indicate the beginning of the interacting fragments. 



SEQUENCE 1. 14-3-3 PROTEIN (X56468) 



1 GTGGTGGGACTCGCGTCGCGGCCGCGGAGACGTGAAGCTCTCGAG 
4 6 GCTCCTCCCGCTGCGGGTCGGCGCTCGCCCTCGCTCTCCTCGCCC 

9 1 TCCGCCCCGGCCCCGGCCCCGGCCCCGCGCCCGCCATGGAGAAGA 

MetGluLysT 

136 CTGAGCTGATCCAGAAGGCCAAGCTGGCCGAGCAGGCCGAGCGCT 
hrGluLeuIleGlnLysAlaLysLeuAlaGluGlnAlaGluArgT 

181 ACGACGACATGGCCACCTGCATGAAGGCAGTGACCGAGCAGGGCG 
yrAspAspMetAlaThrCysMetLysAlaValThrGluGlnGlyA 

▼ 
226 CCGAGCTGTCCAACGAGGAGCGCAACCTGCTCTCCGTGGCCTACA 
laGluLeuSerAsnGluGluArgAsnLeuLeuSerValAlaTyrL 
T T 

271 AGAACGTGGTCGGGGGCCGCAGGTCCGCCTGGAGGGTCATCTCTA 
ysAsnValValGlyGlyArgArgSerAlaTrpArgVallleSerS 

T ▼ 

316 GCATCGAGCAGAAGACCGACACCTCCGACAAGAAGTTGCAGCTGA 
erlleGluGlnLysThrAspThrSerAspLysLysLeuGlnLeuI 

361 TTAAGGACTATCGGGAGAAAGTGGAGTCCGAGCTGAGATCCATCT 
leLysAspTyrArgGluLysValGluSerGluLeuArgSerlleC 

4 06 GCACCACGGTGCTGGAATTGTTGGATAAATATTTAATAGCCAATG 
ysThrThrValLeuGluLeuLeuAspLysTyrLeuIleAlaAsnA 

4 51 CAACTAATCCAGAGAGTAAGGTCTTCTATCTGAAAATGAAGGGTG 
laThrAsnProGluSerLysValPheTyrLeuLysMetLysGlyA 



124 



496 ATTACTTCCGGTACCTTGCTGAAGTTGCGTGTGGTGATGATCGAA 
spTyrPheArgTyrLeuAlaGluValAlaCysGlyAspAspArgL 

541 AACAAACGATAGATAATTCCCAAGGAGCTTACCAAGAGGCATTTG 
ysGlnThrlleAspAsnSerGlnGlyAlaTyrGlnGluAlaPheA 

586 ATATAAGCAAGAAAGAGATGCAACCCACACACCCAATCCGCCTGG 
spIleSerLysLysGluMetGlnProThrHisProIleArgLeuG 

631 GGCTTGCTCTTAACTTTTCTGTATTTTACTATGAGATTCTTAATA 
lyLeuAlaLeuAsnPheSerValPheTyrTyrGluIleLeuAsnA 

676 ACCCAGAGCTTGCCTGCACGCTGGCTAAAACGGCTTTTGATGAGG 
snProGluLeuAlaCysThrLeuAlaLysThrAlaPheAspGluA 

721 CCATTGCTGAACTTGATACACTGAATGAAGACTCATACAAAGACA 
lalleAlaGluLeuAspThrLeuAsnGluAspSerTyrLysAspS 

766 GCACCCTCATCATGCAGTTGCTTAGAGACAACCTAACACTTTGGA 
erThrLeuIleMetGlnLeuLeuArgAspAsnLeuThrLeuTrpT 

811 CATCAGACAGTGCAGGAGAAGAATGTGATGCGGCAGAAGGGGCTG 
hrSerAspSerAlaGlyGluGluCysAspAlaAlaGluGlyAlaG 

856 AAAACTAAATCCATACAGGGTGTCATCCTTCTTTCCTTCAAGAAA 
luAsn 

901 CCTTTTTACACATCTCCATTCCTTATTCCACTTGGATTTCCTATA 

946 GCAAAGAAACCCATTCATGTGTATGGAATCAACTGTTTATAGTCT 

991 TTTCACACTGCAGCTTTGGGAAAACTTCATTCCTTGATTTGTGTT 

1036 TGTCTTGGCCTTCCTGGTGTGCAGTACTGCTGTAGAAAAGTATTA 

1081 ATAGCTTCATTTCATATAAACATAAGTAACTCCCAAACACTTATG 

1126 TAGAGGACTAAAAATGTATCTGGTATTTAAGTAATCTGAACCAGT 

1171 TCTGCAAGTGACTGTGTTTTGTATTACTGTGAAAATAAGAAAATG 

1216 TAGTTAATTACAATTTAAAGAGTATTCCACATAACTTCTTAATTT 

1261 CTACATTCCCTCCCTTACTCTTCGGGGGTTTCCTTTCAGTAAGCA 

13 06 ACTTTTCCATGCTCTTAATGTATTCCTTTTTAGTAGGAATCCGGA 

13 51 AGTATTAGATTGAATGGAAAAGCACTTGCCATCTCTGTCTAGGGG 

13 96 TCACAAATTGAAATGGCTCCTGTATCACATACCGGAGGTCTTGTG 

1441 TATCTGTGGCCAACAGGGAGTTTCCTTATTCACTCTTTATTTGCT 

1486 GCTGTTTAAGTTGCCAACCTCCCCTCCCAATAAAAATTCACTTAC 

1531 ACCTCCTGCCTTTGTAGTTCTGGTATTCACTTTACTATGTGATAG 

1576 AAGTAGCATGTTGCTGCCAGAATACAAGCATTGCTTTTGGCAAAT 

1621 TAAAGTGCATGTCATTTCTTAATACACTAGAAAGGGGAAATAAAT 

1666 TAAAGTACACAAGTCCAAGTCTAAAACTTTAGTACTTTTCCCATG 

1711 CAGATTTGTGCACATGTGAGAGGGTGTCCAGTTTGTCTAGTGATT 

1756 GTTATTTAGAGAGTTGGACCACTATTGTGTGTTGCTAATCATTGA 

18 01 CTGTAGTCCCAAAAAAGCCTTGTGAAAATGTTATGCCCTATGTAA 

1846 CAGCAGAGTAACATAAA 



125 
SEQUENCE 2. Bl 4-3-3 PROTEIN (X57346) 



1 TACCGCCACCGCCGCCGCCGATTCCGGAGCCGGGGTAGTCGCCGC 

4 6 CGCCGCCGCCGCCGCTGCAGCCACTGCAGGCACCGCTGCCGCCGC 

9 1 CTGAGTAGTGTACCGCCACCGCCGCCGCCGATTCCGGAGCCGGGG 

136 TAGTCGCCGCCGCCGCCGCCGCCGCTGCAGCCACTGCAGGCACCG 

181 CTGCCGCCGCCTGAGTAGTGGGCTTAGGAAGGAAGAGGTCATCTC 

226 GCTCGGAGCTTCGCTCGGAAGGGTCTTTGTTCCCTGCAGCCCTCC 

271 CACGGCAGAGTCTCCAGAGATTTGGGCCGCTACAAAAAGTGCATT 

316 TTGCCCATTCGGCTGTGGATAGAGAAGCAGGAAGAGCACTGGACT 

361 TGGAGTCAGGGAATGACAATGGATAAAAGTGAGCTGGTACAGAAA 
MetThrMetAspLysSerGluLeuValGlnLys 

406 GCCAAACTCGCTGAGCAGGCTGAGCGCTATGATGATATGGCTGCA 
AlaLysLeuAlaGluGlnAlaGluArgTyrAspAspMetAlaAla 

T 
4 51 GCCATGAAGGCAGTCACAGAACAGGGGCATGAACTCTCCAACGAA 

AlaMetLysAlaValThrGluGlnGlyHisGluLeuSerAsnGlu 

4 96 GAGAGAAATCTGCTCTCTGTTGCCTACAAGAATGTGGTAGGCGCC 
GluArgAsnLeuLeuSerValAlaTyrLysAsnValValGlyAla 

541 CGCCGCTCTTCCTGGCGTGTCATCTCCAGCATTGAGCAGAAAACA 
ArgArgSerSerTrpArgVallleSerSerlleGluGlnLysThr 

586 GAGAGGAATGAGAAGAAGCAGCAGATGGGCAAAGAGTACCGTGAG 
GluArgAsnGluLysLysGlnGlnMetGlyLysGluTyrArgGlu 

631 AAGATAGAGGCAGAACTGCAGGACATCTGCAATGATGTTCTGGAG 
LysIleGluAlaGluLeuGlnAspIleCysAsnAspValLeuGlu 

6 76 CTGTTGGACAAATATCTTATTCCCAATGCTACACAACCAGAAAGT 
LeuLeuAspLysTyrLeuIleProAsnAlaThrGlnProGluSer 

721 AAGGTGTTCTACTTGAAAATGAAAGGAGATTATTTTAGGTATCTT 
LysValPheTyrLeuLysMetLysGlyAspTyrPheArgTyrLeu 

766 TCTGAAGTGGCATCTGGAGACAACAAACAAACCACTGTGTCGAAC 
SerGluValAlaSerGlyAspAsnLysGlnThrThrValSerAsn 

811 TCCCAGCAGGCTTACCAGGAAGCATTTGAAATTAGTAAGAAAGAA 
SerGlnGlnAlaTyrGlnGluAlaPheGluIleSerLysLysGlu 

856 ATGCAGCCTACACACCCAATTCGTCTTGGTCTGGCACTAAATTTC 
MetGlnProThrHisProIleArgLeuGlyLeuAlaLeuAsnPhe 

901 TCAGTCTTTTACTATGAGATTCTAAACTCTCCTGAAAAGGCCTGT 
SerValPheTyrTyrGluIleLeuAsnSerProGluLysAlaCys 

946 AGCCTGGCAAAAACGGCATTTGATGAAGCAATTGCTGAATTGGAT 
SerLeuAlaLysThrAlaPheAspGluAlalleAlaGluLeuAsp 



126 



991 ACGCTGAATGAAGAGTCTTATAAAGACAGCACTCTGATCATGCAG 
ThrLeuAsnGluGluSerTyrLysAspSerThrLeuIleMetGln 

1036 TTACTTAGGGACAATCTCACTCTGTGGACATCGGAAAACCAGGGA 
LeuLeuArgAspAsnLeuThrLeuTrpThrSerGluAsnGlnGly 

1081 GACGAAGGAGACGCTGGGGAGGGAGAGAACTAATGTTTCTCGTGC 
AspGluGlyAspAlaGlyGluGlyGluAsn 

1126 TTTGTGATCTGTCCAGTGTCACTCTGTACCCTCAACATATATCCC 
1171 TTGTGCGATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 



127 
SEQUENCE 3. CASEIN KINASE II P-SUBUNIT (M30448) 

U + 

1 
TCCGGGCCTGGCCTGTACGGTCGCCGCCGTTCCCTGGAAGTAGCA 

4 6 ACTTCCCTACCC^CCCCAGTCCTGGTCCCCGTCCAWCCGCTGAC 

9 1 GTGAAGATGAGCAGCTCAGAGGAGGTGTCCTGGATTTCCTGGTTC 
MetSerSerSerGluGluValSerTrpIleSerTrpPhe 

136 TGTGGGCTCCGTGGCAATGAATTCTTCTGTGAAGTGGATGAAGAC 
CysGlyLeuArgGlyAsnGluPhePheCysGluValAspGluAsp 

181 TACATCCAGGACAAATTTAATCTTACTGGACTCAATGAGCAGGTC 
TyrlleGlnAspLysPheAsnLeuThrGlyLeuAsnGluGlnVal 

226 CCTCACTACCGACAAGCTCTAGACATGATCTTGGACCTGGAGCCT 
ProHisTyrArgGlnAlaLeuAspMetlleLeuAspLeuGluPro 

2 71 GATGAAGAACTGGAAGACAACCCCAACCAGAGTGACCTGATTGAG 
AspGluGluLeuGluAspAsnProAsnGlnSerAspLeuIleGlu 

316 CAGGCAGCCGAGATGCTTTATGGATTGATCCACGCCCGCTACATC 
GlnAlaAlaGluMetLeuTyrGlyLeuIleHisAlaArgTyrlle 

361 CTTACCAACCGTGGCATCGCCCAGATGTTGGAAAAGTACCAGCAA 
LeuThrAsnArgGlylleAlaGlnMetLeuGluLysTyrGlnGln 

4 06 GGAGACTTTGGTTACTGTCCTCGTGTGTACTGTGAGAACCAGCCA 
GlyAspPheGlyTyrCysProArgValTyrCysGluAsnGlnPro 

451 ATGCTTCCCATTGGCCTTTCAGACATCCCAGGTGAAGCCATGGTG 
MetLeuProIleGlyLeuSerAspIleProGlyGluAlaMetVal 

496 AAGCTCTACTGCCCCAAGTGCATGGATGTGTACACACCCAAGTCA 
LysLeuTyrCysProLysCysMetAspValTyrThrProLysSer 

541 TCAAGACACCATCACACGGATGGCGCCTACTTCGGCACTGGTTTC 
SerArgHisHisHisThrAspGlyAlaTyrPheGlyThrGlyPhe 

586 CCTCACATGCTCTTCATGGTGCATCCCGAGTACCGGCCCAAGAGA 
ProHisMetLeuPheMetValHisProGluTyrArgProLysArg 

631 CCTGCCAACCAGTTTGTGCCCAGGCTCTACGGTTTCAAGATCCAT 
ProAl aAsnGlnPheVa 1 ProArgLeuTyrGlyPheLy s 1 1 eHi s 

6 76 GCGATGGCCTACCAGCTGCAGCTCCAAGCCGCCAGCAACTTCAAG 
AlaMetAlaTyrGlnLeuGlnLeuGlnAlaAlaSerAsnPheLys 

721 AGCCCAGTCAAGACGATTCGCTGATTCCCTCCCCCACCTGTCCTG 
SerProValLysThrlleArg 



128 



766 CAGTCTTTGACTTTTCCTTTCTTTTTTGCCACCCTTTCAGGAACC 

811 CTGTATGGTTTTTAGTTTAAATTAAAGGAGTCGTTATTGTGGTGG 

856 GAATATGAAATAAAGTAGAAGAAAAGGCCATGAAAAAAAAAAAAA 

901 AAAAAAAAAAAAAGGTTTGTGTTCTCGACGCCTTGGTAGTTGGCA 

946 TAGGCTAAAGAAAAGGGATCTCAGCCCCGAGGAAGGGTCACCCTC 

991 CTAGAGATAGCTACTACCCCGTCTCAGGAGACCCTGGTATTTCTA 

1036 GAGCACGCTTTGCTTTCACCAAACCCAAGGAGGTGACAGGAGGAG 

1081 CCCCCGCACAGGACCTAAGAATGCTGTGACCAGAAGATGGGATCG 

1126 CGGAACAGCAGCAGTGCAGGATCCGGTCCGGAGACCCCTCCGAGG 

1171 GCTTGCCCCGAAGAGGGGCTGGCCTGCGTCGGAGTGAGGAAGAGG 

1216 AAGAAGAGGATGAAGATGTGGATCTGGCCCAGGTACTGGCCTATC 

1261 TCCTCCGCAGAGGCCAAGTGAGGTTGGTGCAGGGAGGAGGTGCAG 

13 06 CAAATTTACAATTCATTCAGGCCCTCTTGGACTCAGAGGAAGAGA 

1351 ATGACAGAGCTTGGGATGGTCGTCTTGGGGATCGATACAACCCAC 

13 96 CTGTGGATGCTACCCCTGACACCCGGGAGCTGGAATTCCTGGAGT 

1441 TGTGAACGCCGCGGACTCCGGAGCCGCACAAACCAGGGCTCGCCA 

1486 TGAAGCCAGGATTCAGTCCCCCGTGGGGGTGGCTTTGGCGGCCGA 

1531 GGGGGCTTTGGTGACCGTGGTGGTCGTGGAGGCCGAGGGGGCTTT 

1576 GGCGGGGGCCGAGGTCGAGGCGGAGGCTTTAGAGGTCGTGGACGA 

1621 GGAGGAGGTGGAGGCGGCGGCGGCGGTGGAGGAGGAGGAAGAGGT 

1666 GGAGGCTTCCATTCTGGTGGCAACCGGGGTCGTGGTCGGGGAGGA 

1711 AAAAGAGGAAACCAGTCGGGGAAGAATGTGATGGTGGAGCCGCAT 

1756 CGGACTGAGGGTGTCTTCATTTGTCGAGGAAAGGAGATGCACTGG 

1801 TCACCAAGAACCTGGTCCTGGGAATCAAGTTATGGAGAGAAGAGA 

1846 GTCTCGATTCGGAAGGAGATGACAAAATTGAGTACCGAGCCTGGA 

18 91 ACCCCTTCCGCTCCAAGCTAGCAGCAGCAATCCTGGGTGGTGTGG 

1936 ACCAGATCCACATCAAACCGGGGGCTAAGGTTCTCTACCTCGGGG 

1981 CTGCCTCGGGCACCACGGTCTCCCATGTCTCTGACATCGTTGGTC 

2 026 CGGATGGTCTAGTCTATGCAGTCGAGTTTCCCCACCGCTCTGGCC 

2 071 GTGACCTCATTAACTTGGCCAAGAAGAGGACCAACATCATTCCTG 

2116 TGATCGAGGATGCTCGACACCCACACAAATACCGCATGCTCATCG 

2161 CAATGGTGGATGTGATCTTTGCTGATGTGGCCCAGCCAGACCAGA 

22 06 CCCGGAATGTGGCCCTGAAAGCCCACACCTTCCTGCGCTAATGGA 

2251 GGACACTTTGTGATTTCCATTAAGGCCAACTGCATTGACTCCACA 

22 96 GCCTAGCGGTCAGGCCGTGTTTGCCTCCGAAGTGAAAAAGATGCA 

2341 ACAGGAGAACATGAAGCCGCAGGAGCAGTTGACCCTTGAGCCATA 

2386 TGAAAGAGACCATGCCGTGGTCGTGGGAGTGTACAGGCCACCCCC 

2431 CAAGGTGAAGAACTGAAGTTCAGCGCTGTCAGGATTGCGAGAGAT 

24 76 GTGTGTTGATACTGTTGCCCGTGTGTTTTTCTAATTAAAAGACTC 

2521 CTCCGTC 



129 



SEQUENCE 4. HEPATOCYTE GROWTH FACTOR- 
REGULATED TYROSINE KINASE SUBSTRATE 
(HRS, D84064) 



1 GGGCGCGCCAGCTCGTAGCAGGGGAGCGCCCGCGGCGTCGGGTTT 

4 6 GGGCTGGAGGTCGCCATGGGGCGAGGCAGCGGCACCTTCGAGCGT 

MetGlyArgGlySerGlyThrPheGluArg 

9 1 CTCCTAGACAAGGCGACCAGCCAGCTCCTGTTGGAGACAGATTGG 
LeuLeuAspLysAlaThrSerGlnLeuLeuLeuGluThrAspTrp 

136 GAGTCCATTTTGCAGATCTGCGACCTGATCCGCCAAGGGGACACA 
GluSerlleLeuGlnlleCysAspLeuIleArgGlnGlyAspThr 

181 CAAGCAAAATATGCTGTGAATTCCATCAAGAAGAAAGTCAACGAC 
GlnAlaLysTyrAlaValAsnSerlleLysLysLysValAsnAsp 

226 AAGAACCCACACGTCGCCTTGTATGCCCTGGAGGTCATGGAATCT 
LysAsnProHisValAlaLeuTyrAlaLeuGluValMetGluSer 

T 
2 71 GTGGTAAAGAACTGTGGCCAGACAGTTCATGATGAGGTGGCCAAC 

ValValLysAsnCysGlyGlnThrValHisAspGluValAlaAsn 

316 AAGCAGACCATGGAGGAGCTGAAGGACCTGCTGAAGAGACAAGTG 
LysGlnThrMetGluGluLeuLysAspLeuLeuLysArgGlnVal 

361 GAGGTAAACGTCCGTAACAAGATCCTGTACCTGATCCAGGCCTGG 
GluValAsnValArgAsnLysIleLeuTyrLeuIleGlnAlaTrp 

406 GCGCATGCCTTCCGGAACGAGCCCAAGTACAAGGTGGTCCAGGAC 
AlaHisAlaPheArgAsnGluProLysTyrLysValValGlnAsp 

451 ACCTACCAGATCATGAAGGTGGAGGGGCACGTCTTTCCAGAATTC 
ThrTyrGlnlleMetLysValGluGlyHisValPheProGluPhe 

4 96 AAAGAGAGCGATGCCATGTTTGCTGCCGAGAGAGCCCCAGACTGG 
LysGluSerAspAlaMetPheAlaAlaGluArgAlaProAspTrp 

541 GTGGACGCTGAGGAATGCCACCGCTGCAGGGTGCAGTTCGGGGTG 
ValAspAlaGluGluCysHisArgCysArgValGlnPheGlyVal 

586 ATGACCCGTAAGCACCACTGCCGGGCGTGTGGGCAGATATTCTGT 
MetThrArgLysHisHisCysArgAlaCysGlyGlnllePheCys 

631 GGAAAGTGTTCTTCCAAGTACTCCACCATCCCCAAGTTTGGCATC 
GlyLysCysSerSerLysTyrSerThrlleProLysPheGlylle 

6 76 GAGAAGGAGGTGCGCGTGTGTGAGCCCTGCTACGAGCAGCTGAAC 
GluLysGluValArgValCysGluProCysTyrGluGlnLeuAsn 

721 AGGAAAGCGGAGGGAAAGGCCACTTCCACCACTGAGCTGCCCCCC 
ArgLysAlaGluGlyLysAlaThrSerThrThrGluLeuProPro 



130 



766 GAGTACCTGACCAGCCCCCTGTCTCAGCAGTCCCAGCTGCCCCCC 
GluTyrLeuThrSerProLeuSerGlnGlnSerGlnLeuProPro 

811 AAGAGGGACGAGACGGCCCTGCAGGAGGAGGAGGAGCTGCAGCTG 
LysArgAspGluThrAlaLeuGlnGluGluGluGluLeuGlnLeu 

856 GCCCTGGCGCTGTCACAGTCAGAGGCGGAGGAGAAGGAGAGGCTG 
AlaLeuAlaLeuSerGlnSerGluAlaGluGluLysGluArgLeu 

901 AGACAGAAGTCCACGTACACTTCGTACCCCAAGGCGGAGCCCATG 
ArgGlnLysSerThrTyrThrSerTyrProLysAlaGluProMet 

946 CCCTCGGCCTCCTCAGCGCCCCCCGCCAGCAGCCTGTACTCTTCA 
ProSerAlaSerSerAlaProProAlaSerSerLeuTyrSerSer 

991 CCTGTGAACTCGTCGGCGCCTCTGGCTGAGGACATCGACCCTGAG 
ProValAsnSerSerAlaProLeuAlaGluAspIleAspProGlu 

1036 CTCGCACGGTATCTCAACCGGAACTACTGGGAGAAGAAGCAGGAG 
LeuAlaArgTyrLeuAsnArgAsnTyrTrpGluLysLysGlnGlu 

1081 GAGGCTCGCAAGAGCCCCACGCCATCTGCGCCCGTGCCCCTGACG 
GluAlaArgLysSerProThrProSerAlaProValProLeuThr 

1126 GAGCCGGCTGCACAGCCTGGGGAAGGGCACGCAGCCCCCACCAAC 
GluProAlaAlaGlnProGlyGluGlyHisAlaAlaProThrAsn 

1171 GTGGTGGAGAACCCCCTCCCGGAGACAGACTCTCAGCCCATTCCT 
ValValGluAsnProLeuProGluThrAspSerGlnProIlePro 

1216 CCCTCTGGTGGCCCCTTTAGTGAGCCACAGTTCCACAATGGCGAG 
ProSerGlyGlyProPheSerGluProGlnPheHisAsnGlyGlu 

1261 TCTGAGGAGAGCCACGAGCAGTTCCTGAAGGCGCTGCAGAACGCC 
SerGluGluSerHisGluGlnPheLeuLysAlaLeuGlnAsnAla 

13 06 GTCACCACCTTCGTGAACCGCATGAAGAGTAACCACATGCGGGGC 
ValThrThrPheValAsnArgMetLysSerAsnHisMetArgGly 

13 51 CGCAGCATCACCAATGACTCGGCCGTGCTCTCACTCTTCCAGTCC 
ArgSerlleThrAsnAspSerAlaValLeuSerLeuPheGlnSer 

1396 ATCAACGGCATGCACCCGCAGCTGCTGGAGCTGCTCAACCAGCTG 
IleAsnGlyMetHisProGlnLeuLeuGluLeuLeuAsnGlnLeu 

1441 GACGAGCGCAGGCTGTACTATGAGGGGCTGCAGGACAAGCTGGCA 
AspGluArgArgLeuTyrTyrGluGlyLeuGlnAspLysLeuAla 

1486 CAGATCCGCGATGCCCGGGGGGCGCTGAGTGCCCTGCGCGAAGAG 
GlnlleArgAspAlaArgGlyAlaLeuSerAlaLeuArgGluGlu 

1531 CACCGGGAGAAGCTTCGCCGGGCAGCCGAGGAGGCAGAGCGCCAG 
HisArgGluLysLeuArgArgAlaAlaGluGluAlaGluArgGln 



131 



1576 CGCCAGATCCAGCTGGCCCAGAAGCTGGAGATAATGCGGCAGAAG 
ArgGlnlleGlnLeuAlaGlnLysLeuGluIleMetArgGlnLys 

1621 AAGCAGGAGTACCTGGAGGTGCAGAGGCAGCTGGCCATCCAGCGC 
LysGlnGluTyrLeuGluValGlnArgGlnLeuAlalleGlnArg 

1666 CTGCAGGAGCAGGAGAAGGAGCGGCAGATGCGGCTGGAGCAGCAG 
LeuGlnGluGlnGluLysGluArgGlnMetArgLeuGluGlnGln 

1711 AAGCAGACGGTCCAGATGCGCGCGCAGATGCCCGCCTTCCCCCTG 
LysGlnThrValGlnMetArgAlaGlnMetProAlaPheProLeu 

1756 CCCTACGCCCAGCTCCAGGCCATGCCCGCAGCCGGAGGTGTGCTC 
ProTyrAlaGlnLeuGlnAlaMetProAlaAlaGlyGlyValLeu 

1801 TACCAGCCCTCGGGACCAGCCAGCTTCCCCAGCACCTTCAGCCCT 
TyrGlnProSerGlyProAlaSerPheProSerThrPheSerPro 

1846 GCCGGCTCGGTGGAGGGCTCCCCAATGCACGGCGTGTACATGAGC 
AlaGlySerValGluGlySerProMetHisGlyValTyrMetSer 

18 91 CAGCCGGCCCCTGCCGCTGGCCCCTACCCCAGCATGCCCAGCACT 
GlnProAlaProAlaAlaGlyProTyrProSerMetProSerThr 

1936 GCGGCTGATCCCAGCATGGTGAGTGCCTACATGTACCCAGCAGGG 
AlaAlaAspProSerMetValSerAlaTyrMetTyrProAlaGly 

1981 GCCACTGGGGCGCAGGCGGCCCCCCAGGCCCAGGCCGGACCCACC 
AlaThrGlyAlaGlnAlaAlaProGlnAlaGlnAlaGlyProThr 

2 026 GCCAGCCCCGCTTACTCATCCTACCAGCCTACTCCCACAGCGGGC 
AlaSerProAlaTyrSerSerTyrGlnProThrProThrAlaGly 

2071 TACCAGAACGTGGCCTCCCAGGCCCCACAGAGCCTCCCGGCCATC 
TyrGlnAsnValAlaSerGlnAlaProGlnSerLeuProAlalle 

2116 TCTCAGCCTCCGCAGTCCAGCACCATGGGCTACATGGGGAGCCAG 
SerGlnProProGlnSerSerThrMetGlyTyrMetGlySerGln 

2161 TCAGTCTCCATGGGCTACCAGCCTTACAACATGCAGAATCTCATG 
SerValSerMetGlyTyrGlnProTyrAsnMetGlnAsnLeuMet 

2206 ACCACCCTCCCAAGCCAGGATGCGTCTCTGCCACCCCAGCAGCCC 
ThrThrLeuProSerGlnAspAlaSerLeuProProGlnGlnPro 

22 51 TACATCGCGGGGCAGCAGCCCATGTACCAGCAGATGGCACCCTCT 
TyrlleAlaGlyGlnGlnProMetTyrGlnGlnMetAlaProSer 

22 96 GGCGGTCCCCCCCAGCAGCAGCCCCCCGTGGCCCAGCAACCGCAG 
GlyGlyProProGlnGlnGlnProProValAlaGlnGlnProGln 

2341 GCACAGGGGCCGCCGGCACAGGGCAGCGAGGCCCAGCTCATTTCA 
AlaGlnGlyProProAlaGlnGlySerGluAlaGlnLeuIleSer 



132 



23 86 TTCGACTGACCCAGGCCATGCTCACGTCCGGAGTAACACTACATA 

PheAsp 

2431 CAGTTCACCTGAAACGCCTCGTCTCTAACTGCCGTCGTCCTGCCT 

24 76 CCCTGTCCTCTACTGCCGGTAGTGTCCCTTCTCTGCGAGTGAGGG 
2 521 GGGGCCTTCACCCCAAGCCCACCTCCCTTGTCCTCAGCCTACTGC 
2 566 AGTCCCTGAGTTAGTCTCTGCTTTCTTTCCCCAGGGCTGGGCCAT 
2611 GGGGAGGGAAGGACTTTCTCCCAGGGGAAGCCCCCAGCCCTGTGG 
2656 GTCATGGTCTGTGAGAGGTGGCAGGAATGGGGACCCTCACCCCCC 
2 701 AAGCAGCCTGTGCCCTCTGGCCGCACTGTGAGCTGGCTGTGGTGT 
2 746 CTGGGTGTGGCCTGGGGCTCCCTCTGCAGGGGCCTCTCTCGGCAG 
2 791 CCACAGCCAAGGGTGGAGGCTTCAGGTCTCCAGCTTCTCTGCTTC 
2836 TCAGCTGCCATCTCCAGTGCCCCAGAATGGTACAGCGATAATAAA 
2881 ATGTATTTCAGAAAGG 



133 



SEQUENCE 5. CALMODULIN (D45887) 



1 AGTCCGAGTGGAGAGAGCGAGCTGAGTGGTTGTGTGGTCGCGTCT 

4 6 CGGAAACCGGTAGCGCTTGCAGCATGGCTGACCAACTGACTGAAG 

MetAlaAspGlnLeuThrGluG 

9 1 AGCAGATTGCAGAATTCAAAGAAGCTTTTTCACTATTTGACAAAG 
luGlnlleAlaGluPheLysGluAlaPheSerLeuPheAspLysA 

136 ATGGTGATGGAACTATAACAACAAAGGAATTGGGAACTGTAATGA 
spGlyAspGlyThrlleThrThrLysGluLeuGlyThrValMetA 

181 GATCTCTTGGGCAGAATCCCACAGAAGCAGAGTTACAGGACATGA 
rgSerLeuGlyGlnAsnProThrGluAlaGluLeuGlnAspMetl 

226 TTAATGAAGTAGATGCTGATGGTAATGGCACAATTGACTTCCCTG 
leAsnGluValAspAlaAspGlyAsnGlyThrlleAspPheProG 

2 71 AATTTCTGACAATGATGGCAAGAAAAATGAAAGACACAGACAGTG 
luPheLeuThrMetMetAlaArgLysMetLysAspThrAspSerG 

316 AAGAAGAAATTAGAGAAGCATTCCGTGTGTTTGATAAGGATGGCA 
luGluGluIleArgGluAlaPheArgValPheAspLysAspGlyA 

361 ATGGCTATATTAGTGCTGCAGAACTTCGCCATGTGATGACAAACC 
snGlyTyrlleSerAlaAlaGluLeuArgHisValMetThrAsnL 

406 TTGGAGAGAAGTTAAC AGATGAAGAAGTTGATGAAATGATCAGGG 
euGlyGluLysLeuThrAspGluGluValAspGluMetlleArgG 

451 AAGCAGATATTGATGGTGATGGTCAAGTAAACTATGAAGAGTTTG 
luAlaAspIleAspGlyAspGlyGlnValAsnTyrGluGluPheV 

4 96 TACAAATGATGACAGCAAAGTGAAGACCTTGTACAGAATGTGTTA 

alGlnMetMetThrAlaLys 

541 AATTTCTTGTACAAAATTGTTTATTTGCCTTTTCTTTGTTTGTAA 

586 CTTATCTGTAAAAGGTTTCTCCCTACTGTCAAAAAAATATGCATG 

631 TATAGTAATTAGGACTTCATTCCTCCATGTTTTCTTCCCTTATCT 

6 76 TACTGTCATTGTCCTAAAACCTTATTTTAGAAAATTGATCAAGTA 

721 ACATGTTGCATGTGGCTTACTCTGGATATATCTAAGCCCTTCTGC 

766 ACATCTAAACTTAGATGGAGTTGGTCAAATGAGGGAACATCTGGG 

811 TTATGCCTTTTTTAAAGTAGTTTTCTTTAGGAACTGTCAGCATGT 

8 56 TGTTGTTGAAGTGTGGAGTTGTAACTCTGCGTGGACTATGGACAG 

901 TCAACAATATGTACTTAAAAGTTGCACTATTGCAAAACGGGTGTA 

946 TTATCCAGGTACTCGTACACTATTTTTTTGTACTGCTGGTCCTGT 

991 ACCAGAAACATTTTCTTTTATTGTTACTTGCTTTTTAAACTTTGT 

1036 TTAGCCACTTAAAATCTGCTTATGGCACAATTTGCCTCAAAATCC 

1081 ATTCCAAGTTGTATATTTGTTTTCCAATAAAAAAATTACAATTTA 

1126 CCC 



134 



SEQUENCE 6. ESTROGEN RECEPTOR-RELATED PROTEIN 

(L38487) 



1 TCCTACAAGCAGCCGGCGGCGCCGCCGAGTGAGGGGACGCGGCGC 
SerTyrLysGlnProAlaAlaProProSerGluGlyThrArgArg 

4 6 GGTGGGGCGGCGCGGCCCGAGGAGGCGGCGGAGGAGGGGCCGCCC 
GlyGlyAlaAlaArgProGluGluAlaAlaGluGluGlyProPro 

9 1 GCGGCCCCCGGCTCACTCCGGCACTCCGGGCCGCTCGGCCCCCAT 
AlaAlaProGlySerLeuArgHisSerGlyProLeuGlyProHis 

13 6 GCCTGCCCGACCGCGCTGCCGGAGCCCCAGGTGACCAGCGCCATG 
AlaCysProThrAlaLeuProGluProGlnValThrSerAlaMet 

181 TCCAGCCAGGTGGTGGGCATTGAGCCTCTCTACATCAAGGCAGAG 
SerSerGlnValValGlylleGluProLeuTyrlleLysAlaGlu 

226 CCGGCCAGCCCTGACAGTCCAAAGGGTTCCTCGGAGACAGAGACC 
ProAlaSerProAspSerProLysGlySerSerGluThrGluThr 

271 GAGCCTCCTGTGGCCCTGGCCCCTGGTCCAGCTCCCACTCGCTGC 
GluProProValAlaLeuAlaProGlyProAlaProThrArgCys 

316 CTCCCAGGCCACAAGGAAGAGGAGGATGGGGAGGGGGCTGGGCCT 
LeuProGlyHisLysGluGluGluAspGlyGluGlyAlaGlyPro 

3 61 GGCGAGCAGGGCGGTGGGAAGCTGGTGCTCAGCTCCCTGCCCAAG 

GlyGluGlnGlyGlyGlyLysLeuValLeuSerSerLeuProLys 

4 06 CGCCTCTGCCTGGTCTGTGGGGACGTGGCCTCCGGCTACCACTAT 

ArgLeuCysLeuValCysGlyAspValAlaSerGlyTyrHisTyr 

451 GGTGTGGCATCCTGTGAGGCCTGCAAAGCCTTCTTCAAGAGGACC 
GlyValAlaSerCysGluAlaCysLysAlaPhePheLysArgThr 

496 ATCCAGGGGAGCATCGAGTACAGCTGTCCGGCCTCCAACGAGTGT 
IleGlnGlySerlleGluTyrSerCysProAlaSerAsnGluCys 

541 GAGATCACCAAGCGGAGACGCAAGGCCTGCCAGGCCTGCCGCTTC 
GluIleThrLysArgArgArgLysAlaCysGlnAlaCysArgPhe 

586 ACCAAGTGCCTGCGGGTGGGCATGCTCAAGGAGGGAGTGCGCCTG 
ThrLysCysLeuArgValGlyMetLeuLysGluGlyValArgLeu 

631 GACCGCGTCCGGGGTGGGCGGCAGAAGTACAAGCGGCGGCCGGAG 
AspArgValArgGlyGlyArgGlnLysTyrLysArgArgProGlu 

676 GTGGACCCACTGCCCTTCCCGGGCCCCTTCCCTGCTGGGCCCCTG 
ValAspProLeuProPheProGlyProPheProAlaGlyProLeu 

721 GCAGTCGCTGGAGGCCCCCGGAAGACAGCCCCAGTGAATGCACTG 
AlaValAlaGlyGlyProArgLysThrAlaProValAsnAlaLeu 



135 



766 GTGTCTCATCTGCTGGTGGTTGAGCCTGAGAAGCTCTATGCCATG 
ValSerHisLeuLeuValValGluProGluLysLeuTyrAlaMet 

811 CCTGACCCCGCAGGCCCTGATGGGCACCTCCCAGCCGTGGCTACC 
ProAspProAlaGlyProAspGlyHisLeuProAlaValAlaThr 

856 CTCTGTGACCTCTTTGACCGAGAGATTGTGGTCACCATCAGCTGG 
LeuCysAspLeuPheAspArgGluIleValValThrlleSerTrp 

901 GCCAAGAGCATCCCAGGCTTCTCATCGCTGTCGCTGTCTGACCAG 
AlaLysSerlleProGlyPheSerSerLeuSerLeuSerAspGln 

946 ATGTCAGTACTGCAGAGCGTGTGGATGGAGGTGCTGGTGCTGGGT 
MetSerValLeuGlnSerValTrpMetGluValLeuValLeuGly 

991 GTGGCCCAGCGCTCACTGCCACTGCAGGATGAGCTGGCCTTCGCT 
ValAlaGlnArgSerLeuProLeuGlnAspGluLeuAlaPheAla 

1036 GAGGACTTAGTCCTGGATGAAGAGGGGGCACGGGCAGCTGGCCTG 
GluAspLeuValLeuAspGluGluGlyAlaArgAlaAlaGlyLeu 

1081 GGGGAACTGGGGGCTGCCCTGCTGCAACTAGTGCGGCGGCTGCAG 
GlyGluLeuGlyAlaAlaLeuLeuGlnLeuValArgArgLeuGln 

1126 GCCCTGCGGCTGGAGCGAGAGGAGTATGTTCTACTAAAGGCCTTG 
Al aLeuArgLeuGluArgGluGluTyrVa 1 LeuLeuLys Al aLeu 

1171 GCCCTTGCCAATTCAGACTCTGTGCACATCGAAGATGCCGAGGCT 
AlaLeuAlaAsnSerAspSerValHisIleGluAspAlaGluAla 

1216 GTGGAGCAGCTGCGAGAAGCTCTGCACGAGGCCCTGCTGGAGTAT 
ValGluGlnLeuArgGluAlaLeuHisGluAlaLeuLeuGluTyr 

1261 GAAGCCGGCCGGGCTGGCCCCGGAGGGGGTGCTGAGCGGCGGCGG 
GluAlaGlyArgAlaGlyProGlyGlyGlyAlaGluArgArgArg 

13 06 GCGGGCAGGCTGCTGCTCACGCTACCGCTCCTCCGCCAGACAGCG 
AlaGlyArgLeuLeuLeuThrLeuProLeuLeuArgGlnThrAla 

1351 GGCAAAGTGCTGGCCCATTTCTATGGGGTGAAGCTGGAGGGCAAG 
GlyLysValLeuAlaHisPheTyrGlyValLysLeuGluGlyLys 

13 96 GTGCCCATGCACAAGCTGTTCTTGGAGATGCTCGAGGCCATGATG 
ValProMetHisLysLeuPheLeuGluMetLeuGluAlaMetMet 

1441 GACTGAGGCAAGGGGTGGGACTGGTGGGGGTTCTGGCAGGACCTG 
Asp 

1486 CCTAGCATGGGGTCAGCCCCAAGGGCTGGGGCGGAGCTGGGGTCT 
1531 GGGCAGTGCCACAGCCTGCTGGCAGGGCCAGGGCAATGCCATCAG 
1576 CCCCTGGGAACAGGCCCCACGCCCTCTCCTCCCCCTCCTAGGGGG 
1621 TGTCAGAAGCTGGGAACGTGTGTCCAGGCTCTGGGCACAGTGCTG 
1666 CCCCTTGCAAGCCATAACGTGCCCCCAGAGTGTAGGGGGCCTTGC 



136 



1711 GGAAGCCATAGGGGGCTGCACGGGATGCGTGGGAGGCAGAAACCT 
1756 ATCTCAGGGAGGGAAGGGGATGGAGGCCAGAGTCTCCCAGTGGGT 
18 01 GATGCTTTTGCTGCTGCTTAATCCTACCCCCTCTTCAAAGCAGAG 
1846 TGGGACTTGGAGAGCAAAGGCCCATGCCCCCTTCGCTCCTCCTCT 
18 91 CATCATTTGCATTGGGCATTAGTGTCCCCCCTTGAAGCAATAACT 
1936 CCAAGCAGACTCCAGCCCCTGGACCCCTGGGGTGGCCAGGGCTTC 
1981 CCCATCAGCTCCCAACGAGCCTCCTCAGGGGGTAGGAGAGCACTG 
2 026 CCTCTATGCCCTGCAGAGCAATAACACTATATTTATTTTTGGGTT 
2 071 TGGCCAGGGAGGCGCAGGGACATGGGGCAAGCCAGGGCCCAGAGC 
2116 CCTTGGCTGTACAGAGACTCTATTTTAATGTATATTTGCTGCAAA 
2161 GAGAAACCGCTTTTGGTTTTAAACCTTTAATGAGAAAAAAATATA 
22 06 TAATACCGAGCTC 



137 

SEQUENCE 7. IMPORTIN (3-SUBUNIT (L38951) 



1 CTCCCTCGCTCCCTCCCTGCGCGCCGCCTCTCACTCACAGCCTCC 
4 6 CTTCCTTCTTTCTCCCTCCGCCTCCCGAGCACCAGCGCGCTCTGA 
9 1 GCTGCCCCCAGGGTCCCCTCCCCCGCCGCCAGCAGCCCATTTGGA 
13 6 GGGAGGAAGTAAGGGAAGAGGAGAGGAAGGGGAGCCGGACCGACT 
181 ACCCAGACAGAGCCGGTGAATGGGTTTGTGGTGACCCCCGCCCCC 
22 6 CACCCCACCCTCCCTTCCCACCCGACCCCCAACCCCCATCCCCAG 
2 71 TTCGAGCCGCCGCCCGAAAGGCCGGGCCGTCGTCTTAGGAGGAGT 
316 CGCCGCCGCCGCCACCTCCGCCATGGAGCTGATCACCATTCTCGA 

MetGluLeuIleThrlleLeuGl 

361 GAAGACCGTGTCTCCCGATCGGCTGGAGCTGGAAGCGGCGCAGAA 
uLysThrValSerProAspArgLeuGluLeuGluAlaAlaGlnLy 

406 GTTCCTGGAGCGTGCGGCCGTGGAGAACCTGCCCACTTTCCTTGT 
sPheLeuGluArgAlaAlaValGluAsnLeuProThrPheLeuVa 

4 51 GGAACTGTCCAGAGTGCTGGCAAATCCAGGAAACAGTCAGGTTGC 
lGluLeuSerArgValLeuAlaAsnProGlyAsnSerGlnValAl 

4 96 CAGAGTTGCAGCTGGTCTACAAATCAAGAACTCTTTGACATCTAA 
aArgValAlaAlaGlyLeuGlnlleLysAsnSerLeuThrSerLy 

541 AGATCCAGATATCAAGGCACAATATCAGCAGAGGTGGCTTGCTAT 
sAspProAspIleLysAlaGlnTyrGlnGlnArgTrpLeuAlall 

586 TGATGCTAATGCTCGACGAGAAGTCAAGAACTATGTTTTGCAGAC 
eAspAlaAsnAlaArgArgGluValLysAsnTyrValLeuGlnTh 

631 ATTGGGTACAGAAACTTACCGGCCTAGTTCTGCCTCACAGTGTGT 
rLeuGlyThrGluThrTyrArgProSerSerAlaSerGlnCysVa 

676 GGCTGGTATTGCTTGTGCAGAGATCCCAGTAAACCAGTGGCCAGA 
lAlaGlylleAlaCysAlaGluIleProValAsnGlnTrpProGl 

721 ACTCATTCCTCAGCTGGTGGCCAATGTCACAAACCCCAACAGCAC 
uLeuIleProGlnLeuValAlaAsnValThrAsnProAsnSerTh 

766 AGAGCACATGAAGGAGTCGACATTGGAAGCCATCGGTTATATTTG 
rGluHisMetLysGluSerThrLeuGluAlalleGlyTyrlleCy 

811 CCAAGATATAGACCCAGAGCAGCTACAAGATAAATCCAATGAGAT 
sGlnAspIleAspProGluGlnLeuGlnAspLysSerAsnGluIl 

856 TCTGACTGCCATAATCCAGGGGATGAGGAAAGAAGAGCCTAGTAA 
eLeuThrAlallelleGlnGlyMetArgLysGluGluProSerAs 

901 TAATGTGAAGCTAGCTGCTACAAATGCACTCCTGAACTCATTGGA 
nAsnValLysLeuAlaAlaThrAsnAlaLeuLeuAsnSerLeuGl 

946 GTTCACCAAAGCAAACTTTGATAAAGAGTCTGAAAGGCACTTTAT 
uPheThrLysAlaAsnPheAspLysGluSerGluArgHisPhell 



138 



991 TATGCAGGTGGTCTGTGAAGCCACACAGTGTCCAGATACGAGGGT 
eMetGlnValValCysGluAlaThrGlnCysProAspThrArgVa 

1036 ACGAGTGGCTGCTTTACAGAATCTGGTGAAGATAATGTCCTTATA 
lArgValAlaAlaLeuGlnAsnLeuValLysIleMetSerLeuTy 

1081 TTATCAGTACATGGAGACATATATGGGTCCTGCTCTTTTTGCAAT 
rTyrGlnTyrMetGluThrTyrMetGlyProAlaLeuPheAlall 

1126 CACAATCGAAGCAATGAAAAGTGACATTGATGAGGTGGCTTTACA 
eThrlleGluAlaMetLysSerAspIleAspGluValAlaLeuGl 

1171 AGGGATAGAATTCTGGTCCAATGTCTGTGATGAGGAAATGGATTT 
nGlylleGluPheTrpSerAsnValCysAspGluGluMetAspLe 

1216 GGCCATTGAAGCTTCAGAGGCAGCAGAACAAGGACGGCCCCCTGA 
uAlalleGluAlaSerGluAlaAlaGluGlnGlyArgProProGl 

1261 GCACACCAGCAAGTTTTATGCGAAGGGAGCACTACAGTATCTGGT 
uHisThrSerLysPheTyrAlaLysGlyAlaLeuGlnTyrLeuVa 

1306 TCCAATCCTCACACAGACACTAACTAAACAGGACGAAAATGATGA 
lProIleLeuThrGlnThrLeuThrLysGlnAspGluAsnAspAs 

13 51 TGACGATGACTGGAACCCCTGCAAAGCAGCAGGGGTGTGCCTCAT 
pAspAspAspTrpAsnProCysLysAlaAlaGlyValCysLeuMe 

13 96 GCTTCTGGCCACCTGCTGTGAAGATGACATTGTCCCACATGTCCT 
tLeuLeuAlaThrCysCysGluAspAspIleValProHisValLe 

1441 CCCCTTCATTAAAGAACACATCAAGAACCCAGATTGGCGGTACCG 
uProPhelleLysGluHisIleLysAsnProAspTrpArgTyrAr 

1486 GGATGCAGCAGTGATGGCTTTTGGTTGTATCTTGGAAGGACCAGA 
gAspAlaAlaValMetAlaPheGlyCysIleLeuGluGlyProGl 

1531 GCCCAGTCAGCTCAAACCACTAGTTATACAGGCTATGCCCACCCT 
uProSerGlnLeuLysProLeuVallleGlnAlaMetProThrLe 

1576 AATAGAATTAATGAAAGACCCCAGTGTAGTTGTTCGAGATACAGC 
uIleGluLeuMetLysAspProSerValValValArgAspThrAl 

1621 TGCATGGACTGTAGGCAGAATTTGTGAGCTGCTTCCTGAAGCTGC 
aAlaTrpThrValGlyArglleCysGluLeuLeuProGluAlaAl 

1666 CATCAATGATGTCTACTTGGCTCCCCTGCTACAGTGTCTGATTGA 
a I leAsnAspValTyrLeuAlaProLeuLeuGlnCysLeuI 1 eGl 

1711 GGGTCTCAGTGCTGAACCCAGAGTGGCTTCAAATGTGTGCTGGGC 
uGlyLeuSerAlaGluProArgValAlaSerAsnValCysTrpAl 

1756 TTTCTCCAGTCTGGCTGAAGCTGCTTATGAAGCTGCAGACGTTGC 
aPheSerSerLeuAlaGluAlaAlaTyrGluAlaAlaAspValAl 



139 



18 01 TGATGATCAGGAAGAACCAGCTACTTACTGCTTATCTTCTTCATT 
aAspAspGlnGluGluProAlaThrTyrCysLeuSerSerSerPh 

1846 TGAACTCATAGTTCAGAAGCTCCTAGAGACTACAGACAGACCTGA 
eGluLeuIleValGlnLysLeuLeuGluThrThrAspArgProAs 

1891 TGGACACCAGAACAACCTGAGGAGTTCTGCATATGAATCTCTGAT 
pGlyHisGlnAsnAsnLeuArgSerSerAlaTyrGluSerLeuMe 

1936 GGAAATTGTGAAAAACAGTGCCAAGGATTGTTACCCTGCTGTCCA 
tGluIleValLysAsnSerAlaLysAspCysTyrProAlaValGl 

1981 GAAAACGACTTTGGTCATCATGGAACGACTGCAACAGGTTCTTCA 
nLysThrThrLeuVallleMetGluArgLeuGlnGlnValLeuGl 

2 026 GATGGAGTCACATATCCAGAGCACATCCGATAGAATCCAGTTCAA 
nMetGluSerHisIleGlnSerThrSerAspArglleGlnPheAs 

2071 TGACCTTCAGTCTTTACTCTGTGCAACTCTTCAGAATGTTCTTCG 
nAspLeuGlnSerLeuLeuCysAlaThrLeuGlnAsnValLeuAr 

2116 GAAAGTGCAACATCAAGATGCTTTGCAGATCTCTGATGTGGTTAT 
gLysValGlnHisGlnAspAlaLeuGlnlleSerAspValValMe 

2161 GGCCTCCCTGTTAAGGATGTTCCAAAGCACAGCTGGGTCTGGGGG 
tAlaSerLeuLeuArgMetPheGlnSerThrAlaGlySerGlyGl 

2206 AGTACAAGAGGATGCCCTGATGGCAGTTAGCACACTGGTGGAAGT 
yValGlnGluAspAlaLeuMetAlaValSerThrLeuValGluVa 

2251 GTTGGGTGGTGAATTCCTCAAGTACATGGAGGCCTTTAAACCCTT 
lLeuGlyGlyGluPheLeuLysTyrMetGluAlaPheLysProPh 

2296 CCTGGGCATTGGATTAAAAAATTATGCTGAATACCAGGTTTGTTT 
eLeuGlylleGlyLeuLysAsnTyrAlaGluTyrGlnValCysLe 

2341 GGCAGCTGTGGGCTTAGTGGGAGACTTGTGCCGTGCCCTGCAATC 
uAlaAlaValGlyLeuValGlyAspLeuCysArgAlaLeuGlnSe 

2386 CAACATCATACCTTTCTGTGACGAGGTGATGCAGCTGCTTCTGGA 
rAsnllelleProPheCysAspGluValMetGlnLeuLeuLeuGl 

2431 AAATTTGGGGAATGAGAACGTCCACAGGTCTGTGAAGCCGCAGAT 
uAsnLeuGlyAsnGluAsnValHisArgSerValLysProGlnll 

2476 TCTGTCAGTGTTTGGTGATATTGCCCTTGCTATTGGAGGAGAGTT 
eLeuSerValPheGlyAspIleAlaLeuAlalleGlyGlyGluPh 

2521 TAAAAAATACTTAGAGGTTGTATTGAATACTCTTCAGCAGGCCTC 
eLysLysTyrLeuGluValValLeuAsnThrLeuGlnGlnAlaSe 

2566 CCAAGCCCAGGTGGACAAGTCAGACTATGACATGGTGGATTATCT 
rGlnAlaGlnValAspLysSerAspTyrAspMetValAspTyrLe 



140 



2611 GAATGAGCTAAGGGAAAGCTGCTTGGAAGCCTATACTGGAATCGT 
uAsnGluLeuArgGluSerCysLeuGluAlaTyrThrGlylleVa 

2656 CCAGGGATTAAAGGGGGATCAGGAGAACGTACACCCGGATGTGAT 
lGlnGlyLeuLysGlyAspGlnGluAsnValHisProAspValMe 

2 701 GCTGGTACAACCCAGAGTAGAATTTATTCTGTCTTTCATTGACCA 
tLeuValGlnProArgValGluPhelleLeuSerPhelleAspHi 

2746 CATTGCTGGAGATGAGGATCACACAGATGGAGTAGTAGCTTGTGC 
sIleAlaGlyAspGluAspHisThrAspGlyValValAlaCysAl 

2791 TGCTGGACTAATAGGGGACTTATGTACAGC ATTTGGGAAGGATGT 
aAlaGlyLeuIleGlyAspLeuCysThrAlaPheGlyLysAspVa 

2836 ACTGAAATTAGTAGAAGCTAGGCCAATGATCCATGAATTGTTAAC 
lLeuLysLeuValGluAlaArgProMetlleHisGluLeuLeuTh 

2881 TGAAGGGCGGAGATCGAAGACTAACAAAGCAAAAACCCTTGCTAC 
rGluGlyArgArgSerLysThrAsnLysAlaLysThrLeuAlaTh 

2926 ATGGGCAACAAAAGAACTGAGGAAACTGAAGAACCAAGCTTGATC 
rTrpAlaThrLysGluLeuArgLysLeuLysAsnGlnAla 

2 971 TGTTACCATTGGGATGATAACCTGAGGACCCCCACTGGAAATCTC 

3 016 CCATCTTTTGAAAAACCTGGAAGTGAGGAGTGTGCACGGATGCTG 
3 061 AATGTTTGGGAATGAGAGGATGAGTGAGTGAGGCTTGAAAACACA 
3106 CCACATTGAAAATCCTGCCACAGCAGCAGCCGCAGCCGCCAACAG 
3151 CAGCGCTGTTAGTGAGCTAAGTAAGCACTGACTTCGTAGAAAACC 
3196 ATAACATCGGCCATCTTGGAAAAGAGAAAAACAATGGAGTTACTT 
3241 ATTTAAAAAAAAAGAAAGAAAGTTATCTCTTCCCAGGAGAGGCTA 

32 86 GAAGTAGCTTTTCTGTCTTTTGGCCAGTGCCGAGTGGAATGCCTG 
3331 GTTTCGGGGAGGAGGAGGGACTGGGTTCAGCTGTGGTGCTTTGTT 

33 76 GTAAAAGGCAGCCTGGCCTTTGCTACTGAGGAGAAAGATGGAGCC 
3421 TGGGTCTCAAGCCCACCTTCGCTGTACCTTTGCCACATGGTACTG 
3466 TATGCTTGCCAGCTAGAAGGAGGGTCAGGGTTTTTTACAGTCTGA 
3511 GAATGAGTGTGTGTGAGTGAGGCGGTATCCACATTCTCAACTTCA 
3 556 AGTCATTGCTGTTTCTTTTTCCCAGAAAACAAGGGGTTAGATGTT 
3601 GCATTTCATAAAACTAACCGAAGTTCTGTCTACTGATGCAGCACA 
3646 AGAGATGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACACACA 
3691 AAAAAACACACACACAGAGGAAAGACGCTCTTTAGGTTTTGTTTT 
3 736 GTTTTTTTTTTTTTGGTTTTGTTTTTTGTTTTTTTTTTTACTCTA 
3 781 GGGAAAACACTGACGAATGGTCAGAGCTCCTATCCTGATCTTTTC 
3 826 ATCAAGGCGCCTTTCCTAATAATATGGTTCAACTGTGAATGTAGA 
3871 AGTGGGGGNNAGGGGGGAGAAAAAGAAAACTCTGGCGTTAGAGGA 

3 916 TATAAAAAAATATAAGTACAATTGTTACAAATAACGCAGACTTCA 
3961 AAAACAAAAAAATCACAACCCAAACAAACCAAAATTTAAATGATC 
4006 AGAATTGGCAGCACAAAGAAAACGCCCTCTCCTGACTTGTATTGT 
4051 GGCAGTCTGAACGCCCCCAGAAAATTGTGCCAAAGAGTTTAGAAA 

4 096 AATAAATATAC AATAAAAGTAAACAC ATACACACAAAAC AGCAAA 
4141 CTTCAGGTAACTATTTTGGATTGCAAACAGGATAATTAAATGTTC 
4186 AAACAATCTGATAAAATAACCATTTGG 



141 



SEQUENCE 8. IMOGEN 38 (Z68747) 



1 CGGCGATGTTTCCTAGAGTCTCGACGTTCCTACCTCTTCGCCCCC 
MetPheProArgValSerThrPheLeuProLeuArgProL 

4 6 TTTCCCGCCACCCTTTGTCCTCTGGAAGCCCGGAGACATCAGCGG 
euSerArgHisProLeuSerSerGlySerProGluThrSerAlaA 

9 1 CTGCGATTATGCTACTCACTGTTCGGCACGGAACAGTCAGGTACC 
laAlalleMetLeuLeuThrValArgHisGlyThrValArgTyrA 

136 GCAGTTCAGCGCTGTTGGCCCGGACAAAAAATAACATCCAAAGAT 
rgSerSerAlaLeuLeuAlaArgThrLysAsnAsnlleGlnArgT 

181 ATTTTGGCACTAACAGTGTGATCTGTAGCAAGAAAGATAAGCAGT 
yrPheGlyThrAsnSerVallleCysSerLysLysAspLysGlnS 

226 CTGTTCGAACTGAGGAGACTTCCAAGGAGACTTCAGAGAGCCAAG 
erValArgThrGluGluThrSerLysGluThrSerGluSerGlnA 

2 71 ACAGTGAAAAGGAAAATACGAAAAAAGACTTGTTAGGCATTATTA 

spSerGluLysGluAsnThrLysLysAspLeuLeuGlyllelleL 

316 AGGGCATGAAAGTTGAATTAAGCACAGTAAATGTACGAACAACAA 
ysGlyMetLysValGluLeuSerThrValAsnValArgThrThrL 

3 61 AGCCCCCCAAAAGAAGACCACTTAAAAGTTTGGAAGCTACACTTG 

ysProProLysArgArgProLeuLysSerLeuGluAlaThrLeuG 

4 06 GCAGGCTTCGAAGAGCTACAGAATATGCTCCAAAGAAGAGAATTG 

lyArgLeuArgArgAlaThrGluTyrAlaProLysLysArglleG 

T 
4 51 AGCCCCTGAGTCCTGAGTTGGTGGCAGCTGCATCTGCTGTGGCAG 

luProLeuSerProGluLeuValAlaAlaAlaSerAlaValAlaA 

496 ATTCTCTCCCTTTTGATAAGCAAACAACCAAGTCAGAGCTGCTGA 
spSerLeuProPheAspLysGlnThrThrLysSerGluLeuLeuS 

541 GCCAGCTCCAGCAGCATGAGGAAGAGTCAAGGGCACAGAGAGATG 
erGlnLeuGlnGlnHisGluGluGluSerArgAlaGlnArgAspA 

586 CAAAGCGACCTAAAATTAGTTTCAGTAACATAATATCAGATATGA 
laLysArgProLysIleSerPheSerAsnllelleSerAspMetL 

631 AAGTTGCCAGATCTGCTACAGCTAGAGTTCGTTCAAGACCAGAGC 
ysValAlaArgSerAlaThrAlaArgValArgSerArgProGluL 

676 TTCGGATTCAGTTTGATGAAGGCTATGACAATTATCCTGGCCAGG 
euArglleGlnPheAspGluGlyTyrAspAsnTyrProGlyGlnG 

721 AGAAGACGGATGATCTTAAAAAAAGGAAAAATATATTCACAGGGA 
luLysThrAspAspLeuLysLysArgLysAsnllePheThrGlyL 



142 



766 AAAGACTTAATATTTTTGACATGATGGCAGTTACTAAAGAAGCAC 
ysArgLeuAsnllePheAspMetMetAlaValThrLysGluAlaP 

811 CTGAAACAGACACATCACCTTCACTTTGGAATGTGGAATTTGCTA 
roGluThrAspThrSerProSerLeuTrpAsnValGluPheAlaL 

856 AGCAGTTAGCCACAGTAAATGAACAACCCCTTCAGAATGGATTTG 
YsGlnLeuAlaThrValAsnGluGlnProLeuGlnAsnGlyPheG 

901 AAGAGCTGATCCAGTGGACAAAAGAGGGGAAACTATGGGAGTTCC 
luGluLeuIleGlnTrpThrLysGluGlyLysLeuTrpGluPheP 

946 CAATTAACAATGAAGCAGGTTTTGATGATGATGGTTCAGAATTTC 
roIleAsnAsnGluAlaGlyPheAspAspAspGlySerGluPheH 

991 ATGAACATATATTTCTGGAGAAACACCTGGAGAGCTTTCCAAAAC 
isGluHisIlePheLeuGluLysHisLeuGluSerPheProLysG 

1036 AAGGACCAATTCGCCACTTCATGGAGCTGGTGACTTGTGGCCTTT 
InGlyProIleArgHisPheMetGluLeuValThrCysGlyLeuS 

1081 CCAAAAACCCATATCTTAGTGTTAAACAGAAGGTTGAACACATAG 
erLysAsnProTyrLeuSerValLysGlnLysValGluHisIleG 

1126 AGTGGTTTAGAAATTATTTTAATGAAAAAAAGGATATTCTAAAAG 
luTrpPheArgAsnTyrPheAsnGluLysLysAspIleLeuLysG 

1171 AAAGTAACATACAGTTCAATTAAGACCATGGA 
luSerAsnlleGlnPheAsn 



143 
SEQUENCE 9. DGS-I (DIGEORGE SYNDROME, L77566) 



1 GGGGATAGCGATGGAGACGCCGGGCGCATCAGCGTCGTCCTTGTT 
GlylleAlaMetGluThrProGlyAlaSerAlaSerSerLeuLe 

4 6 GCTTCCCGCCGCGTCCAGGCCCCCGAGGAAGCGCGAGGCGGGAGA 
uLeuProAlaAlaSerArgProProArgLysArgGluAlaGlyGl 

9 1 GGCTGGGGCTGCGACGAGCAAGCAGCGGGTCCTGGACGAGGAAGA 
uAlaGlyAlaAlaThrSerLysGlnArgValLeuAspGluGluGl 

136 GTATATCGAGGGCCTCCAGACGGTCATCCAAAGGGATTTCTTTCC 
uTyrlleGluGlyLeuGlnThrVallleGlnArgAspPhePhePr 

181 TGATGTGGAGAAGCTCCAGGCACAGAAGGAGTACCTGGAAGCCGA 
oAspValGluLysLeuGlnAlaGlnLysGluTyrLeuGluAlaGl 

226 GGAGAATGGAGACTTGGAACGGATGCGCCAGATTGCCATCAAGTT 
uGluAsnGlyAspLeuGluArgMetArgGlnlleAlalleLysPh 

2 71 TGGCTCTGCCTTGGGCAAGATGTCCCGGGAGCCCCCGCCACCCTA 

eGlySerAlaLeuGlyLysMetSerArgGluProProProProTy 

316 TGTGACTCCAGCCACATTTGAAACCCCTGAGGTGCATGCAGGCAC 
rValThrProAlaThrPheGluThrProGluValHisAlaGlyTh 

3 61 TGGAGTGGTGGGCAACAAGCCCAGGCCCCGCGGCCGAGGCCTGGA 

rGlyValValGlyAsnLysProArgProArgGlyArgGlyLeuGl 

4 06 GGATGGAGAGGCTGGAGAGGAGGAGGAGAAGGAGCCGCTGCCCAG 

uAspGlyGluAlaGlyGluGluGluGluLysGluProLeuProSe 

4 51 CCTAGATGTCTTCCTGAGCCGCTACACGAGTGAGGACAATGCCTC 
rLeuAspValPheLeuSerArgTyrThrSerGluAspAsnAlaSe 

496 CTTCCAGGAGATCATGGAGGTGGCCAAGGAGAGAAGCCGGGCACG 
rPheGlnGluIleMetGluValAlaLysGluArgSerArgAlaAr 

541 CCACGCTTGGCTCTACCAGGCTGAGGAAGAGTTTGAGAAGAGGCA 
gHisAlaTrpLeuTyrGlnAlaGluGluGluPheGluLysArgGl 

586 GAAAGATAATCTCGAACTCCCGTCAGCAGAGCACCAGGCCATCGA 
nLysAspAsnLeuGluLeuProSerAlaGluHisGlnAlalleGl 

631 GAGCAGCCAGGCCAGTGTGGAGACCTGGAAGTACAAGGCCAAGAA 
uSerSerGlnAlaSerValGluThrTrpLysTyrLysAlaLysAs 

676 TTCCCTCATGTACTATCCAGAGGGTGTCCCTGACGAGGAGCAGCT 
nSerLeuMetTyrTyrProGluGlyValProAspGluGluGlnLe 

721 GTTTAAGAAGCCCCGGCAGGTGGTACATAAGAACACGCGCTTCCT 
uPheLysLysProArgGlnValValHisLysAsnThrArgPheLe 



144 



766 TAGGGACCCCTTCAGCCAAGCCCTGAGCAGGTGCCAGCTCCAGCA 
uArgAspProPheSerGlnAlaLeuSerArgCysGlnLeuGlnGl 

811 GGCAGCCGCCCTCAATGCCCAGCACAAACAGGGCAAGGTGGGCCC 
nAlaAlaAlaLeuAsnAlaGlnHisLysGlnGlyLysValGlyPr 

8 56 CGATGGCAAGGAGCTGATCCCCCAGGAGTCCCCTCGAGTGGGTGG 
oAspGlyLysGluLeuIleProGlnGluSerProArgValGlyGl 

901 ATTTGGATTTGTTGCCACTCCTTCCCCTGCCCCTGGTGTGAACGA 
yPheGlyPheValAlaThrProSerProAlaProGlyValAsnGl 

946 GTCCCCGATGATGACCTGGGGGGAGGTTGAGAACACACCCTTGAG 
uSerProMetMetThrTrpGlyGluValGluAsnThrProLeuAr 

991 AGTTGAAGGGTCGGAAACGCCCTACGTGGACAGGACACCCGGCCC 
gValGluGlySerGluThrProTyrValAspArgThrProGlyPr 

1036 AGCTTTTAAGATCCTGGAGCCAGGCCGCAGGGAGCGGCTGGGTCT 
oAlaPheLysIleLeuGluProGlyArgArgGluArgLeuGlyLe 

1081 GAAGATGGCCAACGAGGCCGCTGCCAAGAACCGGGCCAAGAAGCA 
uLysMetAlaAsnGluAlaAlaAlaLysAsnArgAlaLysLysGl 

1126 GGAAGCCTTGCGGAGAGTGACGGAGAATCTGGCCAGCCTCACCCC 
nGluAlaLeuArgArgValThrGluAsnLeuAlaSerLeuThrPr 

1171 CAAAGGCCTGAGCCCAGCCATGTCGCCAGCCCTACAGCGCCTTGT 
oLysGlyLeuSerProAlaMetSerProAlaLeuGlnArgLeuVa 

1216 GAGCAGGACGGCCAGCAAGTACACAGACCGGGCCCTGCGGGCCAG 
lSerArgThrAlaSerLysTyrThrAspArgAlaLeuArgAlaSe 

1261 CTACACACCATCCCCAGCACGCTCCACCCACCTCAAGACCCCGGC 
rTyrThrProSerProAlaArgSerThrHisLeuLysThrProAl 

13 06 CAGTGGGCTGCAGACCCCCACAAGCACACCGGCGCCTGGCTCTGC 
aSerGlyLeuGlnThrProThrSerThrProAlaProGlySerAl 

1351 CACACGCACCCCTCTCACACAGGACCCGGCCTCCATCACGGACAA 
aThrArgThrProLeuThrGlnAspProAlaSerlleThrAspAs 

13 96 CCTGCTGCAGCTCCCTGCCCGGCGCAAAGCTTCGGACTTCTTTTA 

nLeuLeuGlnLeuProAlaArgArgLysAlaSerAspPhePhe 
1441 GAGCCAGGCCTGGGCTGGGCTCATAGACGCTTCACAGAGCCTGCA 

14 86 GGGCAGCTGTACACCCAGCAGAGGACTCCAGCCTTCTCGGGGCCC 
1531 AGGCCTGGGCCAGAAGCTGTTGACCATACCAGGAGTCACTGGAGA 
1576 AAGGGGCTGTGCTGGGGCCAGACTGGCACAAGGCACTCGTGCCCA 
1621 CACCACACCCCAGGGCCTTGCCAAGCTGTTTGCTGTTTAATTGGC 
1666 CCCTTGAACTGTCATTAAAGAACACCTAGGTAC 



145 



SEQUENCE 10. ATROPHIN 1 (U23851) 



1 TTGGGGTGGAGCAGAGAAGTTTCTGTATTCAGCTGCCCAGGCAGA 

4 6 GGAGAATGGGGTCTCCACAGCCTGAAGAATGAAGACACGACAGAA 

MetLysThrArgGlnAs 

9 1 TAAAGACTCGATGTCAATGAGGAGTGGACGGAAGAAAGAGGCCCC 
nLysAspSerMetSerMetArgSerGlyArgLysLysGluAlaPr 

13 6 TGGGCCCCGGGAAGAACTGAGATCGAGGGGCCGGGCCTCCCCTGG 
oGlyProArgGluGluLeuArgSerArgGlyArgAlaSerProGl 

181 AGGGGTCAGCACGTCCAGCAGTGATGGCAAAGCTGAGAAGTCCAG 
yGlyValSerThrSerSerSerAspGlyLysAlaGluLysSerAr 

226 GCAGACAGCCAAGAAGGCCCGAGTAGAGGAAGCCTCCACCCCAAA 
gGlnThrAlaLysLysAlaArgValGluGluAlaSerThrProLy 

2 71 GGTCAACAAGCAGGGTCGGAGTGAGGAGATCTCAGAGAGTGAAAG 

sValAsnLysGlnGlyArgSerGluGluIleSerGluSerGluSe 

316 TGAGGAGACCAATGCACCAAAAAAGACCAAAACTGAGGAACTCCC 
rGluGluThrAsnAlaProLysLysThrLysThrGluGluLeuPr 

3 61 TCGGCCACAGTCTCCCTCCGATCTGGATAGCTTGGACGGGCGGAG 

oArgProGlnSerProSerAspLeuAspSerLeuAspGlyArgSe 

4 06 CCTTAATGATGATGGCAGCAGCGACCCTAGGGATATCGACCAGGA 

rLeuAsnAspAspGlySerSerAspProArgAspIleAspGlnAs 

451 CAACCGAAGCACGTCCCCCAGTATCTACAGCCCTGGAAGTGTGGA 
pAsnArgSerThrSerProSerlleTyrSerProGlySerValGl 

496 GAATGACTCTGACTCATCTTCTGGCCTGTCCCAGGGCCCAGCCCG 
uAsnAspSerAspSerSerSerGlyLeuSerGlnGlyProAlaAr 

541 CCCCTACCACCCACCTCCACTCTTTCCTCCTTCCCCTCAACCGCC 
gProTyrHisProProProLeuPheProProSerProGlnProPr 

586 AGACAGCACCCCTCGACAGCCAGAGGCTAGCTTTGAACCCCATCC 
oAspSerThrProArgGlnProGluAlaSerPheGluProHisPr 

631 TTCTGTGACACCCACTGGATATCATGCTCCCATGGAGCCCCCCAC 
oSerValThrProThrGlyTyrHisAlaProMetGluProProTh 

676 ATCTCGAATGTTCCAGGCTCCTCCTGGGGCCCCTCCCCCTCACCC 
rSerArgMetPheGlnAlaProProGlyAlaProProProHisPr 

721 ACAGCTCTATCCCGGGGGCACTGGTGGAGTTTTGTCTGGACCCCC 
oGlnLeuTyrProGlyGlyThrGlyGlyValLeuSerGlyProPr 



146 



766 AATGGGTCCCAAGGGGGGAGGGGCTGCCTCATCAGTGGGGGGCCC 
oMetGlyProLysGlyGlyGlyAlaAlaSerSerValGlyGlyPr 

811 TAATGGGGGTAAGCAGCACCCCCCACCCACTACTCCCATTTCAGT 
oAsnGlyGlyLysGlnHisProProProThrThrProIleSerVa 

856 ATCAAGCTCTGGGGCTAGTGGTGCTCCCCCAACAAAGCCGCCTAC 
lSerSerSerGlyAlaSerGlyAlaProProThrLysProProTh 

901 CACTCCAGTGGGTGGTGGGAACCTACCTTCTGCTCCACCACCAGC 
rThrProValGlyGlyGlyAsnLeuProSerAlaProProProAl 

946 CAACTTCCCCCATGTGACACCGAACCTGCCTCCCCCACCTGCCCT 
aAsnPheProHisValThrProAsnLeuProProProProAlaLe 

991 GAGACCCCTCAACAATGCATCAGCCTCTCCCCCTGGCCTGGGGGC 
uArgProLeuAsnAsnAlaSerAlaSerProProGlyLeuGlyAl 

1036 CCAACCACTACCTGGTCATCTGCCCTCTCCCCACGCCATGGGACA 
aGlnProLeuProGlyHisLeuProSerProHisAlaMetGlyGl 

1081 GGGTATCGGTGGACTTCCTCCTGGCCCAGAGAAGGGCCCAACTCT 
nGlylleGlyGlyLeuProProGlyProGluLysGlyProThrLe 

1126 GGCTCCTTCACCCCACTCTCTGCCTCCTGCTTCCTCTTCTGCTCC 
uAlaProSerProHisSerLeuProProAlaSerSerSerAlaPr 

1171 AGCGCCCCCCATGAGGTTTCCTTATTCATCCTCTAGTAGTAGCTC 
oAlaProProMetArgPheProTyrSerSerSerSerSerSerSe 

1216 TGCAGCAGCCTCCTCTTCCAGTTCTTCCTCCTCTTCCTCTGCCTC 
TAlaAlaAlaSerSerSerSerSerSerSerSerSerSerAlaSe 

1261 CCCCTTCCCAGCTTCCCAGGCATTGCCCAGCTACCCCCACTCTTT 
rProPheProAlaSerGlnAlaLeuProSerTyrProHisSerPh 

13 06 CCCTCCCCCAACAAGCCTCTCTGTCTCCAATCAGCCCCCCAAGTA 
eProProProThrSerLeuSerValSerAsnGlnProProLysTy 

13 51 TACTCAGCCTTCTCTCCCATCCCAGGCTGTGTGGAGCCAGGGTCC 
rThrGlnProSerLeuProSerGlnAlaValTrpSerGlnGlyPr 

1396 CCCACCACCTCCTCCCTATGGCCGCCTCTTAGCCAACAGCAATGC 
oProProProProProTyrGlyArgLeuLeuAlaAsnSerAsnAl 

1441 CCATCCAGGCCCCTTCCCTCCCTCTACTGGGGCCCAGTCCACCGC 
aHisProGlyProPheProProSerThrGlyAlaGlnSerThrAl 

1486 CCACCCACCAGTCTCAACACATCACCATCACCACCAGCAACAGCA 
aHisProProValSerThrHisHisHisHisHisGlnGlnGlnGl 

1531 ACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCATCACGGAAACTC 
nGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnHisHisGlyAsnSe 



147 



1576 TGGGCCCCCTCCTCCTGGAGCATTTCCCCACCCACTGGAGGGCGG 
rGlyProProProProGlyAlaPheProHisProLeuGluGlyGl 

1621 TAGCTCCCACCACGCACACCCTTACGCCATGTCTCCCTCCCTGGG 
ySerSerHisHisAlaHisProTyrAlaMetSerProSerLeuGl 

1666 GTCTCTGAGGCCCTACCCACCAGGGCCAGCACACCTGCCCCCACC 
ySerLeuArgProTyrProProGlyProAlaHisLeuProProPr 

1711 TCACAGCCAGGTGTCCTACAGCCAAGCAGGCCCCAATGGCCCTCC 
oHisSerGlnValSerTyrSerGlnAlaGlyProAsnGlyProPr 

1756 AGTCTCTTCCTCTTCCAACTCTTCCTCTTCCACTTCTCAAGGGTC 
oValSerSerSerSerAsnSerSerSerSerThrSerGlnGlySe 

18 01 CTACCCATGTTCACACCCCTCCCCTTCCCAGGGCCCTCAAGGGGC 
rTyrProCysSerHisProSerProSerGlnGlyProGlnGlyAl 

1846 GCCCTACCCTTTCCCACCGGTGCCTACGGTCACCACCTCTTCGGC 
aProTyrProPheProProValProThrValThrThrSerSerAl 

18 91 TACCCTTTCCACGGTCATTGCCACCGTGGCTTCCTCGCCAGCAGG 
aThrLeuSerThrVallleAlaThrValAlaSerSerProAlaGl 

1936 CTACAAAACGGCCTCCCCACCTGGGCCCCCACCGTACGGAAAGAG 
yTyrLysThrAlaSerProProGlyProProProTyrGlyLysAr 

1981 AGCCCCGTCCCCGGGGGCCTACAAGACAGCCACCCCACCCGGATA 
gAlaProSerProGlyAlaTyrLysThrAlaThrProProGlyTy 

2026 CAAACCCGGGTCGCCTCCCTCCTTCCGAACGGGGACCCCACCGGG 
rLysProGlySerProProSerPheArgThrGlyThrProProGl 

2 071 CTATCGAGGAACCTCGCCACCTGCAGGCCCAGGGACCTTCAAGCC 
yTyrArgGlyThrSerProProAlaGlyProGlyThrPheLysPr 

2116 GGGCTCGCCCACCGTGGGACCTGGGCCCCTGCCACCTGCGGGGCC 
oGlySerProThrValGlyProGlyProLeuProProAlaGlyPr 

2161 CTCAGGCCTGCCATCGCTGCCACCACCACCTGCGGCCCCTGCCTC 
oSerGlyLeuProSerLeuProProProProAlaAlaProAlaSe 

22 06 AGGGCCGCCCCTGAGCGCCACGCAGATCAAACAGGAGCCGGCTGA 
rGlyProProLeuSerAlaThrGlnlleLysGlnGluProAlaGl 

2251 GGAGTATGAGACCCCCGAGAGCCCGGTGCCCCCAGCCCGCAGCCC 
uGluTyrGluThrProGluSerProValProProAlaArgSerPr 

22 96 CTCGCCCCCTCCCAAGGTGGTAGATGTACCCAGCCATGCCAGTCA 
oSerProProProLysValValAspValProSerHisAlaSerGl 

2341 GTCTGCCAGGTTCAACAAACACCTGGATCGCGGCTTCAACTCGTG 
nSerAlaArgPheAsnLysHisLeuAspArgGlyPheAsnSerCy 



148 



2386 CGCGCGCAGCGACCTGTACTTCGTGCCACTGGAGGGCTCCAAGCT 
sAlaArgSerAspLeuTyrPheValProLeuGluGlySerLysLe 

2431 GGCCAAGAAGCGGGCCGACCTGGTGGAGAAGGTGCGGCGCGAGGC 
uAlaLysLysArgAlaAspLeuValGluLysValArgArgGluAl 

2476 CGAGCAGCGCGCGCGCGAAGAAAAGGAGCGCGAGCGCGAGCGGGA 
aGluGlnArgAlaArgGluGluLysGluArgGluArgGluArgGl 

2521 ACGCGAGAAAGAGCGCGAGCGCGAGAAGGAGCGCGAGCTTGAACG 
uArgGluLysGluArgGluArgGluLysGluArgGluLeuGluAr 

2566 CAGCGTGAAGTTGGCTCAGGAGGGCCGTGCTCCGGTGGAATGCCC 
gSerValLysLeuAlaGlnGluGlyArgAlaProValGluCysPr 

2611 ATCTCTGGGCCCAGTGCCCCATCGCCCTCCATTTGAACCGGGCAG 
oSerLeuGlyProValProHisArgProProPheGluProGlySe 
T 

2656 TGCGGTGGCTACAGTGCCCCCCTACCTGGGTCCTGACACTCCAGC 
rAlaValAlaThrValProProTyrLeuGlyProAspThrProAl 

2701 CTTGCGCACTCTCAGTGAATATGCCCGGCCTCATGTCATGTCTCC 
aLeuArgThrLeuSerGluTyrAlaArgProHisValMetSerPr 

2 746 TGGCAATCGCAACCATCCATTCTACGTGCCCCTGGGGGCAGTGGA 
oGlyAsnArgAsnHisProPheTyrValProLeuGlyAlaValAs 

2 791 CCCGGGGCTCCTGGGTTACAATGTCCCGGCCCTGTACAGCAGTGA 
pProGlyLeuLeuGlyTyrAsnValProAlaLeuTyrSerSerAs 

2836 TCCAGCTGCCCGGGAGAGGGAACGGGAAGCCCGTGAACGAGACCT 
pProAlaAlaArgGluArgGluArgGluAlaArgGluArgAspLe 

2881 CCGTGACCGCCTCAAGCCTGGCTTTGAGGTGAAGCCTAGTGAGCT 
uArgAspArgLeuLys ProGlyPheGluVal Lys ProS e rGluLe 

2 926 GGAACCCCTACATGGGGTCCCTGGGCCGGGCTTGGATCCCTTTCC 

uGluProLeuHisGlyValProGlyProGlyLeuAspProPhePr 

2971 CCGACATGGGGGCCTGGCTCTGCAGCCTGGCCCACCTGGCCTGCA 
oArgHisGlyGlyLeuAlaLeuGlnProGlyProProGlyLeuHi 

3 016 CCCTTTCCCCTTTCATCCGAGCCTGGGGCCCCTGGAGCGAGAACG 

sProPheProPheHisProSerLeuGlyProLeuGluArgGluAr 

3 061 TCTAGCGCTGGCAGCTGGGCCAGCCCTGCGGCCTGACATGTCCTA 
gLeuAlaLeuAlaAlaGlyProAlaLeuArgProAspMetSerTy 

3106 TGCTGAGCGGCTGGCAGCTGAGAGGCAGCACGCAGAAAGGGTGGC 
rAlaGluArgLeuAlaAlaGluArgGlnHisAlaGluArgValAl 

3151 GGCCCTGGGCAATGACCCACTGGCCCGGCTGCAGATGCTCAATGT 
aAlaLeuGlyAsnAspProLeuAlaArgLeuGlnMetLeuAsnVa 



149 



3196 GACTCCCCATCACCACCAGCACTCCCACATCCACTCGCACCTGCA 
lThrProHisHisHisGlnHisSerHisIleHisSerHisLeuHi 

3241 CCTGCACCAGCAAGATGCTATCCATGCAGCCTCTGCCTCGGTGCA 
sLeuHisGlnGlnAspAlalleHisAlaAlaSerAlaSerValHi 

3286 CCCTCTCATTGACCCCCTGGCCTCAGGGTCTCACCTTACCCGGAT 
sProLeuIleAspProLeuAlaSerGlySerHisLeuThr Argil 

33 31 CCCCTACCCAGCTGGAACTCTCCCTAACCCCCTGCTTCCTCACCC 
eProTyrProAlaGlyThrLeuProAsnProLeuLeuProHisPr 

3 3 76 TCTGCACGAGAACGAAGTTCTTCGTCACCAGCTCTTTGCTGCCCC 
oLeuHisGluAsnGluValLeuArgHisGlnLeuPheAlaAlaPr 

3421 TTACCGGGACCTGCCGGCCTCCCTTTCTGCCCCGATGTCAGCAGC 
oTyrArgAspLeuProAlaSerLeuSerAlaProMetSerAlaAl 

3466 TCATCAGCTGCAGGCCATGCACGCACAGTCAGCTGAGCTGCAGCG 
aHisGlnLeuGlnAlaMetHisAlaGlnSerAlaGluLeuGlnAr 

3511 CTTGGCGCTGGAACAGCAGCAGTGGCTGCATGCCCATCACCCGCT 
gLeuAlaLeuGluGlnGlnGlnTrpLeuHisAlaHisHisProLe 

3 556 GCACAGTGTGCCGCTGCCTGCCCAGGAGGACTACTACAGTCACCT 
uHisSerValProLeuProAlaGlnGluAspTyrTyrSerHisLe 

3601 GAAGAAGGAAAGCGACAAGCCACTGTAGAACCTGCGATCAAGAGA 
uLysLysGluSerAspLysProLeu 

3 646 GCACCATGGCTCCTACATTGGACCTTGGAGCACCCCCACCCTCCC 

3691 CCCACCGTGCCCTTGGCCTGCCACCCAGAGCCAAGAGGGTGCTGC 

3 736 TCAGTTGCAGGGCCTCCGCAGCTGGACAGAGAGTGGGGGAGGGAG 

3 781 GGACAGACAGAAGGCCAAGGCCCGATGTGGTGTGCAGAGGTGGGG 

3 826 AGGTGGCGAGGATGGGGACAGAAAGCGCACAGAATCTTGGACCAG 

3 871 GTCTCTCTTCCTTGTCCCCCCTGCTTTTCTCCTCCCCCATGCCCA 

3 916 ACCCCTGTGGCCGCCGCCCCTCCCCTGCCCCGTTGGTGTGATTAT 

3 961 TTCATCTGTTAGATGTGGCTGTTTTGCGTAGCATCGTGTGCCACC 

4 006 CCTGCCCCTCCCCGATCCCTGTGTGCGCGCCCCCTCTGCAATGTA 
4051 TGCCCCTTGCCCCTTCCCCACACTAATAATTTATATATATAAATA 
4 096 TCTATATGACGCTCTTAAAAAAACATCCCAACCAAAACCAACCAA 
4141 ACAAAAACATCCTCACAACTCCCCAGGA 



150 



SEQUENCE 11. EMBRYONAL FYN SUBSTRATE 
(EFS2, AB001467) 



1 CTCCAGGCAACTTGGGGCAAGCGTCTCAGTTCTCGCTCTCCCTTC 

4 6 CTCCCAGCGGGGTCGCCGCAGACCCCAGCCCTGGGAGCACCGCTC 

9 1 TGCAGCGCGGCCGGCGGGTGGAGACGGTTGGCCCCTAAACTCGCT 

13 6 CGTCCAGCCCAACCGCCCCGGCGGCTTCTCCCAGCCCTCGAGGCT 

181 CTCCTGAGCGGCCTGGAGAGGCGTCGAGCGCAGCCCAGCGCCCGC 

225 CTGCTCACCCGCCCCGGCCCGGGAAGGGAATTTTCGGATCCTGCG 

2 71 AGCCCGGGGCGCCCCCGCGGCCTAGGGCGGGCAGCTCCCGGGGCC 
316 TGGCCGAGCCGGTGGCGCCCGGGAGGCCGCGGGGACAGCACGCAG 

3 61 CGCGCGCCCTTGGATGCCGTCCCGCAGCGACGCCCCGGCCCGCCC 
406 CGCTCCTCCTCCTGCCTGGCTAGCCTGCCTCTCATTTGGGAAGTT 
451 TTGTGGGTTTTCTTTCTCCTCCTCCAACCTTGGCGGAGGCCACGA 
496 CTCAGGCGCCACAGCTGGGGGCTAGAGGCCGCGGACCATGGTGCG 
541 GGGCAGCCACCGCTGAAGTCAGCAAAACCGAGCCTGGCCTGAGGC 

586 AGGCTGCGCGGGAGGCCAAAGCCATGGCCATTGCCACGTCGACCC 

MetAlalleAlaThrSerThrG 

631 AGCTGGCCCGGGCACTGTATGACAACACCGCTGAGTCCCCCCAGG 
InLeuAlaArgAlaLeuTyrAspAsnThrAlaGluSerProGInG 

6 76 AGCTGTCCTTCCGCCGAGGGGATGTCCTACGGGTCCTGCAGAGAG 
luLeuSerPheArgArgGlyAspValLeuArgValLeuGlnArgG 

721 AGGGCGCTGGTGGACTGGACGGCTGGTGCCTCTGCTCCCTACACG 
luGlyAlaGlyGlyLeuAspGlyTrpCysLeuCysSerLeuHisG 

766 GCCAGCAGGGCATTGTGCCCGCCAACAGGGTGAAGCTCTTGCCTG 
lyGlnGlnGlylleValProAlaAsnArgValLysLeuLeuProA 

811 CTGGCCCAGCACCCAAGCCCAGCCTCTCTCCTGCGTCCCCAGCCC 
laGlyProAlaProLysProSerLeuSerProAlaSerProAlaG 

856 AGCCTGGCTCACCATATCCAGCCCCAGATCACAGCAATGAGGACC 
InProGlySerProTyrProAlaProAspHisSerAsnGluAspG 

901 AGGAGGTGTATGTGGTGCCGCCCCCAGCTCGGCCCTGTCCAACCT 
InGluValTyrValValProProProAlaArgProCysProThrS 

94 6 CAGGACCTCCAGCTGGACCTTGCCCACCCTCTCCTGACCTCATCT 
erGlyProProAlaGlyProCysProProSerProAspLeuIleT 

991 ACAAAATCCCCAGAGCTAGTGGGACCCAGCTGGCTGCTCCCAGAG 
yrLysIleProArgAlaSerGlyThrGlnLeuAlaAlaProArgA 

103 6 ATGCCTTGGAGGTCTACGATGTGCCCCCCACCGCCCTCCGGGTGC 
spAlaLeuGluValTyrAspValProProThrAlaLeuArgValP 

1081 CCTCCAGTGGCCCCTATGACTGCCCTGCCTCCTTTTCCCACCCTC 
roSerSerGlyProTyrAspCysProAlaSerPheSerHisProL 



151 



1126 TGACCCGGGTTGCCCCGCAGCCCCCTGGAGAGGATGATGCTCCCT 
euThrArgValAlaProGlnProProGlyGluAspAspAlaProT 

1171 ATGATGTGCCTCTGACCCCAAAGCCACCTGCAGAGCTGGAACCAG 
yrAspValProLeuThrProLysProProAlaGluLeuGluProA 

1216 ATCTGGAGTGGGAAGGAGGCCGGGAGCCGGGGCCCCCCATCTATG 
spLeuGluTrpGluGlyGlyArgGluProGlyProProIleTyrA 

1261 CTGCCCCCTCCAACCTGAAACGAGCGTCAGCCTTACTCAATTTGT 
laAlaProSerAsnLeuLysArgAlaSerAlaLeuLeuAsnLeuT 

13 06 ATGAAGCACCCGAGGAACTGCTGGCAGACGGGGAGGGCGGGGGCA 
yrGluAlaProGluGluLeuLeuAlaAspGlyGluGlyGlyGlyT 

13 51 CTGATGAGGGGATCTACGATGTGCCTCTGCTGGGGCCAGAGGCTC 
hrAspGluGlylleTyrAspValProLeuLeuGlyProGluAlaP 

13 96 CCCCTTCTCCAGAGCCCCCTGGAGCCTTGGCCTCCCATGACCAGG 
roProSerProGluProProGlyAlaLeuAlaSerHisAspGlnA 

1441 ACACCCTGGCCCAGCTTCTGGCCAGAAGCCCCCCACCCCCACACA 
spThrLeuAlaGlnLeuLeuAlaArgSerProProProProHisA 

1486 GGCCCCGGCTCCCCTCAGCTGAGAGCCTGTCCCGCCGCCCTCTGC 
rgProArgLeuProSerAlaGluSerLeuSerArgArgProLeuP 

1531 CTGCCCTGCCTGTCCCTGAGGCCCCCAGCCCCTCCCCAGTGCCCT 
roAlaLeuProValProGluAlaProSerProSerProValProS 

1576 CTCCTGCCCCAGGCCGGAAGGGCAGCATCCAGGACCGGCCTCTGC 
erProAlaProGlyArgLysGlySerlleGlnAspArgProLeuP 

T 

1621 CCCCACCCCCACCCCGCCTGCCTGGTTATGGAGGCCCCAAGGTCG 

roProProProProArgLeuProGlyTyrGlyGlyProLysValG 

1666 AGGGGGATCCAGAGGGCAGGGAGATGGAGGATGACCCAGCAGGAC 
luGlyAspProGluGlyArgGluMetGluAspAspProAlaGlyH 

1711 ACCACAATGAGTACGAGGGCATTCCGATGGCCGAGGAGTATGACT 
isHisAsnGluTyrGluGlylleProMetAlaGluGluTyrAspT 

1756 ATGTCCACCTGAAGGGCATGGACAAAGCTCAGGGATCTAGGCCCC 
yrValHisLeuLysGlyMetAspLysAlaGlnGlySerArgProP 

18 01 CGGATCAGGCCTGCACAGGGGATCCTGAACTGCCCGAGAGGGGGA 
roAspGlnAlaCysThrGlyAspProGluLeuProGluArgGlyM 

1846 TGCCGGCGCCGCAGGAGGCCCTGTCCCCAGGGGAGCCACTGGTTG 
etProAlaProGlnGluAlaLeuSerProGlyGluProLeuValV 

1891 TGTCCACCGGAGATCTGCAGCTCCTGTACTTCTATGCTGGGCAAT 
alSerThrGlyAspLeuGlnLeuLeuTyrPheTyrAlaGlyGlnC 



152 



1936 GCCAGAGCCACTACTCAGCCCTGCAGGCAGCCGTGGCAGCCCTGA 
ysGlnSerHisTyrSerAlaLeuGlnAlaAlaValAlaAlaLeuM 

1981 TGTCCAGTACCCAGGCTAATCAGCCCCCGCGCCTTTTCGTGCCCC 
etSerSerThrGlnAlaAsnGlnProProArgLeuPheValProH 

2026 ACAGCAAGAGGGTGGTGGTGGCTGCTCATCGCCTGGTGTTTGTTG 
isSerLysArgValValValAlaAlaHisArgLeuValPheValG 

2 071 GGGACACCCTGGGCCGGCTGGCAGCCTCTGCCCCTCTGAGAGCAC 
lyAspThrLeuGlyArgLeuAlaAlaSerAlaProLeuArgAlaG 

2116 AGGTCAGGGCTGCAGGTACAGCACTGGGCCAGGCATTGCGGGCCA 
InValArgAlaAlaGlyThrAlaLeuGlyGlnAlaLeuArgAlaT 

2161 CTGTGCTGGCTGTCAAGGGAGCTGCCCTGGGCTACCCATCCAGCC 
hrValLeuAlaValLysGlyAlaAlaLeuGlyTyrProSerSerP 

2206 CTGCCATCCAAGAGATGGTGCAGTGTGTAACAGAACTGGCAGGGC 
roAlalleGlnGluMetValGlnCysValThrGluLeuAlaGlyG 

2251 AGGCCCTGCAATTCACTACCCTGCTCACTAGCCTGGCTCCATGAA 
InAlaLeuGlnPheThrThrLeuLeuThrSerLeuAlaPro 

22 96 GGTCCTTTGGCACAGCTCTGCTCCTCCCCTGCCTGCCAAAGCCCC 

2341 CCTTTAGGCCTTGGGTGGCTGGAAGGCTTTGTTAAGGGACTAGGA 

2386 GAAATGGGGGTATCTTTCCCCTTTCCTGCCCTTTCTGCTCATCTC 

2431 AACCTCTCACAGAGGTGTCTTCTCCCCCTAACCTACAGCTTTTTG 

24 76 TACAAGCCATTTTGTGTAAATTATTTATATTTAATATTATTCCCT 

2521 GCTTTGTCAGGAGCAGGTACTAGGCTCTGGGGCAGTGAGGAACTA 

2566 GATCCTTCTCTCCTCAGCCTAGGGTGGAGGTCACTGCACTACCAC 

2611 CCACCTCTGGAAGACTGGCTGTGAAAAGTCAGGTGGCAGAAACCT 

2656 GGGGCCACATAGAGCCTCTCTCTTTTCCTGTTTCTTGGCTCTAGA 

2701 AGATCAGCACTGCACTGTTAGCTGAGAGTGCGGGCAAGACATAAA 

2746 CTGTCCAGAGTTTGAAGGTTCTCGGAAAGACCGGAGGGCTTCTCC 

2 791 CCACAGAAGGCGGAGAGAGCTGGGGCTCAGACATGGGTGTGCACC 

2836 TTAATAAACCCTGCTGTCTGCCTCCCTGACTCTGCTTCTTGGGAG 

2881 CATGGTGAGCAGCCCTGGTGCTCAGCAGCCATACCTATGGGACAC 

2926 ACACTACGAAAAGGATGCCTTTAGGGTTTGGGGGAGATTTTACTC 

2 971 CTTTCTTCAACAACTATTCACTGGACAAGTTCTCTGCTCCCATGA 

3 016 CGCGCCAGGCACAGTTCTGCAAGTATATTGTGAATGTATTGTTCT 
3061 AGTGGGATACACAAATAAGTCAGTTAAAATACATAAATAAAAACA 
3106 TAAACCTGC 



153 



SEQUENCE 12. HUMAN BRAIN FACTOR 2 
(HBF-G2, X78202) 



1 GGCGAGAGAGACGCTCCCGCTCGCCGCCAGCTCTGATTGGCCCAG 

4 6 CGGTAGGAAAGGTTAAACCAAAAATTTTTTTACAGCCCTAGTGTG 

9 1 CGCCTGTAGCTCGGAAAATTAATTGTGGCTATAGCCGCCTCGATC 

136 GCTGTCTCCCCAGCCTCGCCGCGGACGCTCCGGGACGCGCCCGCC 

181 CGCCGCCCGGTTCTCCCCCCCTTTGGGCTGGTGCTGCTGCTGCTG 

226 TGACTGCTGCTGCGAAAGGAGGAGGAGGAGGAGGAAGCAGCGGGG 

2 71 GGGGGAGCGGTGGGTGTGGGGGAAACCAAGAGTACAGTGGACGAG 

316 GACTCACCCCGGCGTGGTGTTCTTTTTTCTTCTTCTTTTTCTTTC 

361 CTTTTTTTTTTTTTTTTCTAATTCCTGAGGGGTGGTTGCTGCTTT 

4 06 TGCTACATGACTTGCCAGCGCCCGAGCCTGCGGTCCAACTGCGCT 

451 GCTGCCGGAGCGCTCAGTGCCGCCGCTGCCGCCCGTGCCCCCCGC 

496 GCCCCGTTCGGCACCCACCGGTCGCCGCCCCGCCCGCGCGCCGCT 

541 GTCCCGCTCCCGCGCCGCCGCCGCCGTTTCCCCCCGACGACTGGG 

586 TGATGCTGGACATGGGAGATAGGAAAGAGGTGAAAATGATCCCCA 
MetLeuAspMetGlyAspArgLysGluValLysMetlleProL 

631 AGTCCTCGTTCAGCATCAACAGCCTGGTGCCCGAGGGCCTCCAGA 
ysSerSerPheSerlleAsnSerLeuValProGluGlyLeuGlnA 

676 ACGACAACCACCACGCGAGCCACGGCCACCACAACAGCCACCACC 
snAspAsnHisHisAlaSerHisGlyHisHisAsnSerHisHisP 

721 CCCAGCACCACCACCACCACCACCACCATCACCACCACCCGCCGC 
roGlnHisHisHisHisHisHisHisHisHisHisHisProProP 

766 CGCCCGCCCCGCAACCGCCGCCGCCGCCGCAGCAGCAGCAGCCGC 
roProAlaProGlnProProProProProGlnGlnGlnGlnProP 

811 CGCCGCCGCCGAGACGCGGGGCCCGGCGCCGACGACGACGAGGCC 
roProProProArgArgGlyAlaArgArgArgArgArgArgGlyP 

856 CCAGCAGTTGTTGTTCCGCCGCGCACGCACACGGCGCGCCTGAGG 
roSerSerCysCysSerAlaAlaHisAlaHisGlyAlaProGluG 

901 GCCAACGGCAGCTGGCGCAAGGCGACCGGCGCGGCCGGGGGATCT 
lyGlnArgGlnLeuAlaGlnGlyAspArgArgGlyArgGlylleC 

946 GCCCCGTCGGGCCGGACGAGAAGGAGAAGGCCCGCGCCGGGGGGG 
ysProValGlyProAspGluLysGluLysAlaArgAlaGlyGlyG 

991 AGGAGAAGAAGGGGGCGGGCGAGGGCGGCAAGGACGGGGAGGGGG 
luGluLysLysGlyAlaGlyGluGlyGlyLysAspGlyGluGlyG 

1036 GCAAGGAGGGCGAGAAGAAGAACGGCAAGTACGAGAAGCCGCCGT 
lyLysGluGlyGluLysLysAsnGlyLysTyrGluLysProProP 

1081 TCAGCTACAACGCGCTCATCATGATGGCCATGCGGCAGAGCCCCG 
heSerTyrAsnAlaLeuIleMetMetAlaMetArgGlnSerProG 



154 



1126 AGAAGCGGCTCACGCTCAACGGCATCTACGAGTTCATCATGAAGA 
luLysArgLeuThrLeuAsnGlylleTyrGluPhelleMetLysA 

1171 ACTTCCCTTACTACCGCGAGAACAAGCAGGGCTGGCAGAACTCCA 
snPheProTyrTyrArgGluAsnLysGlnGlyTrpGlnAsnSerl 

1216 TCCGCCACAATCTGTCCCTCAACAAGTGCTTCGTGAAGGTGCCGC 
leArgHisAsnLeuSerLeuAsnLysCysPheValLysValProA 

1261 GCCACTACGACGACCCGGGCAAGGGCAACTACTGGATGCTGGACC 
rgHisTyrAspAspProGlyLysGlyAsnTyrTrpMetLeuAspP 

13 06 CGTCGAGCGACGACGTGTTCATCGGCGGCACCACGGGCAAGCTGC 
roSerSerAspAspValPhelleGlyGlyThrThrGlyLysLeuA 

13 51 GGCGCTCCACCACCTCGCCGGCCAAGCCGGCCTTCAAGCGCGGTG 
rgArgSerThrThrSerProAlaLysProAlaPheLysArgGlyA 

13 96 CCGCGCTCACCTCCACCGGCCTCACCTTCATGGACGCGCCGGCTC 
laAlaLeuThrSerThrGlyLeuThrPheMetAspAlaProAlaP 

1441 CCTCTACTGGCCCATGTCGCCCTTCCTGTCCCTGCACCACCCCCG 
roSerThrGlyProCysArgProSerCysProCysThrThrProA 

1486 CCAGCAGCACTTTGAGTTACAACGGGACCACGTCGGCCTACCCCA 
laSerSerThrLeuSerTyrAsnGlyThrThrSerAlaTyrProS 

1531 GCCACCCCATGCCCTACAGCTCCGTGTTGACTCAAAACTCGCTGG 
erHisProMetProTyrSerSerValLeuThrGlnAsnSerLeuG 

1576 GCAACAACCACTCCTCCTCCACCGCCAACGGGCTGAGCGTGGACC 
lyAsnAsnHisSerSerSerThrAlaAsnGlyLeuSerValAspA 
T 

1621 GGCTGGTCAACGGGGGAATCCCGTACGCCACGCACCACCTCACGG 
rgLeuValAsnGlyGlylleProTyrAlaThrHisHisLeuThrA 

1666 CCGCCGCGCTAACCGCCTCGGTGCCCTGCGGCCTGCTGGTGCCCT 
laAlaAlaLeuThrAlaSerValProCysGlyLeuLeuValProC 

1711 GCTCTGGGACCTACTCCCTCAACCCCTGCTCCGTCAACCTGCTCG 
ysSerGlyThrTyrSerLeuAsnProCysSerValAsnLeuLeuA 

1756 CGGGCCAGACCAGTTACTTTTTCCCCCACGTCCCGCACCCGTCAA 
laGlyGlnThrSerTyrPhePheProHisValProHisProSerM 

1801 TGACTTCGCAGAGCAGCACGTCCATGAGCGCCAGGGCCGCGTCCT 
etThrSerGlnSerSerThrSerMetSerAlaArgAlaAlaSerS 

1846 CCTCCACGTCGCCGGCAGGCCCCCCTCGACCCCTGCCCTGTGAGT 
erSerThrSerProAlaGlyProProArgProLeuProCysGluS 

18 91 CTTTAAGACCCTCTTTGCCAAGTTTTACGACGGGACTGTCTGGGG 
erLeuArgProSerLeuProSerPheThrThrGlyLeuSerGlyG 



155 



1936 GACTGTCTGATTATTTCACACATCAAAATCAGGGGTCTTCTTCCA 
lyLeuSerAspTyrPheThrHisGlnAsnGlnGlySerSerSerA 

1981 ACCCTTTAATACATTAACATCCCTGGGACCAGACTGTAAGTGAAC 
snProLeuIleHis 

2026 GTTTTACAC AC ATTTGCATTGTAAATGATAATTAAAAAAATAAGT 

2 071 CCAGGTATTTTTTATTAAGCCCCCCCCTCCCATTTCTGTACGTTT 

2116 GTTCAGTCTCTAGGGTTGTTTATTATTCTAACAAGGTGTGGAGTG 

2161 TCAGCGAGGTGCAATGTGGGGAGAATACATTGTAGAATATAAGGT 

2206 TTGGAAGTCAAATTATAGTAGAATGTGTATCTAAATAGTGACTGC 

2251 TTTGCCATTTCATTCAAACCTGACAAGTCTATCTCTAAGAGCCGC 

22 96 CAGATTTCCATGTGTGCAGTATTATAAGTTATCATGGAACTATAT 

2341 GGTGGACGCAGACCTTGAGAACAACCTAAATTATGGGGAGAATTT 

2386 TAAAATGTTAAACTGTAATTTGTATTTAAAAAGCATTCGTAGTAA 

2431 AGGTGCCCAAGAAATTATTTTGGCCATTTATTGTTTTCTCCTTTT 

2476 CTTTAAAGAACTGTTTTTTTTTCTTTTGTTTACTTTTAGACCAAA 

2 521 GATTGGGCGGTTCTAGAAAATGCGCCTTGGTATACTAAGTATTAA 

2566 AACAAACAAAAAGGAAAGTTGTTTCAGTTAACGCTGCCCATTCAA 

2611 TTGAATCAGAAGGGGACAAAATTAACGATTGCCTTCAGTTTGTGT 

2656 TGTGTATATTTTGATGTATGTGGTCACTAACAGGTCACTTTTATT 

2701 TTTTCTAAATGTAGTGAAATGTTAATACCTATTGTACTTATAGGT 

2746 AAACCTTGCAAATATGTAACCTGTGTTGCGCAAATGCCGCATAAA 

2791 TTTGAGTGATTGTTAATGTTGTCTTAAAATTTCTTGATTGTGACT 

2836 ATGTGGTCATATGCCCGTGTTTGTCACTTACAAAAATGTTTACTA 

2881 TGAACACACATAAATAAAAAATAG 



156 
SEQUENCE 13. IP-1 



1 GAATTCGGACGAGGGAGGAGGAAGACCAAGACATACAGGGAGAAA 
IleArgThrArgGluGluGluAspGlnAspIleGlnGlyGluI 

4 6 TCAGTCATCCTGATGGAAAGGTGGAAAAGGTTTATAAGAATGGGT 
leSerHisProAspGlyLysValGluLysValTyrLysAsnGlyC 

9 1 GCCGTGTTATACTGTTTCCCAATGGAACTCGAAAGGAAGTGAGTG 
ysArgVallleLeuPheProAsnGlyThrArgLysGluValSerA 

136 CAGATGGGAAGACCATCACTGTCACTTTCTTTAATGGTGACGTGA 
laAspGlyLysThrlleThrValThrPhePheAsnGlyAspValL 

181 AGCAGGTCATGCCAGACCAAAGAGTGATCTACTACTATGCAGCTG 
ysGlnValMetProAspGlnArgVallleTyrTyrTyrAlaAlaA 

226 CCCAGACCACTCACAGGACATACCCGGAGANCTGGGAAGTCTTAC 
laGlnThrThrHisArgThrTyrProGlu TrpGluValLeuH 

271 ATTTCTCAAGTGGACAAATAGAAAAACATTACCCAGATGGAAGAA 
isPheSerSerGlyGlnlleGluLysHisTyrProAspGlyArgL 

316 AAGAAATCACGTTTCCTGACCAGACTGTTAAAAACTTATTTCCTG 
ysGluIleThrPheProAspGlnThrValLysAsnLeuPheProA 

361 ATGGACAAGAAGAAAGCATTTCCCAGATGGTACAATTGTCAGAGT 
spGlyGlnGluGluSerlleSerGlnMetValGlnLeuSerGluT 

406 ACAACCCCCTCGCAACAAACTCATATAGTTTATTATTGACAAAAA 
yrAsnProLeuAlaThrAsnSerTyrSerLeuLeuLeuThrLysL 

451 AAACTAACTACATACTGCCCAGTTCAAGAGACGGGAATACCAGAT 
ysThrAsnTyrlleLeuProSerSerArgAspGlyAsnThrArgT 

496 GGCACTGT 
rpHisCys 



157 
SEQUENCE 14. IP-2 

I 

1 GTGGGAAACATGCGGCTGTCGGTCGCTGCAGCATCTCCCATGGCC 
GlyLysHisAlaAlaValGlyArgCysSerlleSerHisGlyA 

4 6 GCGTATTTCGCCGTATGGGCCTCGGTCCCGAGTCCCGCATCCATC 
rgValPheArgArgMetGlyLeuGlyProGluSerArglleHisL 

9 1 TGTTGCGGAACTTGCTCACAGGGCTGGTGCGGCACGAACGCATCG 
euLeuArgAsnLeuLeuThrGlyLeuValArgHisGluArglleG 

136 AGGCACCATGGGCGCGTGTGGACGAAATGAGGGGCTACGCGGAGA 
luAlaProTrpAlaArgValAspGluMetArgGlyTyrAlaGluL 

181 AGCTCATCGACTATGGGAAGCTGGGAGACACTAACGAACGAGCCA 
ysLeuIleAspTyrGlyLysLeuGlyAspThrAsnGluArgAlaM 

226 TGCGCATGGCTGACTTCTGGCTCACAGAGAAGGATTTGATCCCAA 
etArgMetAlaAspPheTrpLeuThrGluLysAspLeuIleProL 

2 71 AGCTGTTTCAAGTACTGGCCCCTCGGTACAAAGATCAAACTGGGG 
ysLeuPheGlnValLeuAlaProArgTyrLysAspGlnThrGlyG 

316 GCTACACAAGAATGCTGCAGATCCCAAATCGGAGTTTGGATCGGG 
lyTyrThrArgMetLeuGlnlleProAsnArgSerLeuAspArgA 

361 CCAAGATGGCAGTGATCGAGTATAAAGGGAATTGCCTCCCACCCC 
laLysMetAlaVallleGluTyrLysGlyAsnCysLeuProProL 

406 TTGCCTCTTGCCTCCGCAGAGACAAGCCACCTTACACTCCTAAAC 
euAlaSerCysLeuArgArgAspLysProProTyrThrProLysP 

451 CAGCTGCTGCAGGGTTTGCGGCAGGACCTTCAGGCAAAGCCAGGA 
roAlaAlaAlaGlyPheAlaAlaGlyProSerGlyLysAlaArg 



158 
SEQUENCE 15. IP-3 



1 AATAGACCAGGCTTGGGCCAGAATGAGAATCTGAGTGCCATTGAG 
AsnArgProGlyLeuGlyGlnAsnGluAsnLeuSerAlalleGlu 

4 6 GGGAAAGGCAAGGTGGGGGGACTGAAGACACGCTGCTCTAGCTGC 
GlyLysGlyLysValGlyGlyLeuLysThrArgCysSerSerCys 

9 1 AACGTTAAGTTTGAGTCTGAAAGTGAACTCCAGAACCACATCCAA 
AsnValLysPheGluSerGluSerGluLeuGlnAsnHisIleGln 

136 ACCATCCACCGAGAGCTCGTGCCAGACAGCAACAGCACACAGTTG 
ThrlleHisArgGluLeuValProAspSerAsnSerThrGlnLeu 

181 AAAACGCCCCAAGTATCACCAATGCCCAGAATCAGTCCCTCCCAG 
LysThrProGlnValSerProMetProArglleSerProSerGln 

226 TCGGATGAGAAGAAGACCTATCAATGCATCAAGTGTCAGATGGTT 
SerAspGluLysLysThrTyrGlnCysIleLysCysGlnMetVal 

2 71 TTCTACAATGAATGGGATATTCAGGTTCATGTTGCAAATCACATG 
PheTyrAsnGluTrpAspIleGlnValHisValAlaAsnHisMet 



sVa 



316 ATTGATGAAGGACTGAACCATGAATGCAAACTCTGCAGCCAGACC 
IleAspGluGlyLeuAsnHisGluCysLysLeuCysSerGlnThr 

361 TTTGACTCTCCTGCCAAACTCCAGTGCCACCTGATAGAGCACAGC 
PheAspSerProAlaLysLeuGlnCysHisLeuIleGluHisSer 

4 06 TTCGAAGGGATGGGAGGCACCTTCAAGTGTCCAGTCTGCTTTACA 
PheGluGlyMetGlyGlyThrPheLysCysProValCysPheThr 

451 GTATTTGTTCAAGCAAACAAGTTGCAGCAGCATATTTTCTCTGCC 
ValPheValGlnAlaAsnLysLeuGlnGlnHisIlePheSerAla 

496 CATGGACAAGAAGACAAGATCTATGACTGTACACAATGTCCACAG 
HisGlyGlnGluAspLysIleTyrAspCysThrGlnCysProGln 

541 AAGTTTTTCTTCCAAACAGAGCTGCAGAATCATACAATGACCCAA 
LysPhePhePheGlnThrGluLeuGlnAsnHisThrMetThrGln 

586 CACAGCAGTTAGTGCAAGTACAGTCTCTCAAGGAGAATTGATTTT 
HisSerSer 

631 GTGGCACAAAAAGGGAACATGTTTTACTCTTTGCACGAAACTTTC 

6 76 ATTGTTAATGTATATTATTCAGAAACATTGTATTGTACCATAAAA 

721 CTTGTATTATCAAACTGTTGGATGTTCATGTGTTTGAACTTTTGC 

766 GCACCGGATAGACCCCTTGTATATAAAGTGTTGCACATGTATTAT 

811 GTCGTCTGATACTAAAATGGTCTTATAAAGACAAGTGGACTTGGG 

8 56 CCCTATTCAGGCAAGATTAAAAAAAAAAAAAAGACTATGACCAAA 

901 ATGGCTTAAGATAAAGTATTTTTAAGGAAGAAAGATTAAAAACAA 

946 CTGTTATACATGAGACTATGGTTGGACTTCCTTTTCTTTACACTT 

991 AAGCCTAGAATTTCTCTTTAGGTATATCAGCGCTTAAATCCAAGA 



159 



1036 CTATTTTTTATTGCTGAAGATTCTTGCAAACCATGAAGAGATGTT 

1081 CTCACAGAACAGAACCCCCACAGCTGGNATAAGGCCCGGTATATA 

1126 TATATTTGGTAAAGCCCTTGCAATGTGACAGGGTAGCATCACTAA 

1171 TATATGCAATAGGTTGTTATGGAGACTG 



160 
SEQUENCE 16. IP-4 

I 

1 CCTGGCCCAGTACCTCATCACTGCCTGTCTAGAGACGGCCAAGTC 
LeuAlaGlnTyrLeuIleThrAlaCysLeuGluThrAlaLysSe 

4 6 TACCTCTGGGGGGACGCCCAGCTCGGCCCCCGCAGACCTGCCTGC 
rThrSerGlyGlyThrProSerSerAlaProAlaAspLeuProAl 

9 1 CCCCTTCTCGCCACCCCTGACGGCCCTGCCACAGCTCCCCCTGGC 
aProPheSerProProLeuThrAlaLeuProGlnLeuProLeuAl 

136 CTGCTGGGCACACCCTATGCCATCTCCCTCTCCAACTTCATCGGC 
aCysTrpAlaHisProMetProSerProSerProThrSerSerAl 

181 CTCAAGCCCATGCCCTTCTTGGCTTTACCACCTGCTTCCCCAGGG 
aSerSerProCysProSerTrpLeuTyrHisLeuLeuProGlnGl 

226 CCGCCGCCGGGCTTGGCGCTAACACTGCCAAGATGGCAGCAGCTA 
yArgArgArgAlaTrpArg 

271 ATGGGAGCAAGAAGGCTGAGCGGCAGAAATTCTCCCCCTACTGAG 
316 GCCAGCTGAGGTACAGGCAGGGGCAGGCAGGACCACCAGCAGGGG 
361 GCTTGCCTCTGCACCCTACCCGCCCAAGGAGACTCCACCCTGGGG 
406 TCCCAAACGCCGCTAACGCCCAGACGCATGGATTGCACCCCCTAC 
451 CCTGCCTCCATCTATGGGAGTTCTTTCTCTCAGAGTGGGGGCAGT 
496 TTCTGGGCCCAGGGGTCTGAGCTGCGGCA 



161 
SEQUENCE 17. IP-5 



1 GTAGAGATGGCCGCGCTTGCACCGCTGCCCCCGCTCCCCGCACAG 
ValGluMetAlaAlaLeuAlaProLeuProProLeuProAlaGln 

4 6 TTCAAGAGCATACAGCATCATCTGAGGACGGCTCAGGAGCATGAC 
PheLysSerlleGlnHisHisLeuArgThrAlaGlnGluHisAsp 

9 1 AAGCGAGACCCTGTGGTGGCTTATTACTGTCGTTTATACGCAATG 
LysArgAspProValValAlaTyrTyrCysArgLeuTyrAlaMet 

136 CAGACTGGAATGAAGATCGATAGTAAAACTCCTGAATGTCGCAAA 
GlnThrGlyMetLysIleAspSerLysThrProGluCysArgLys 

181 TTTTTATCAAAGTTAATGGATCAGTTAGAAGCTCTAAAGAAGCAG 
PheLeuSerLysLeuMetAspGlnLeuGluAlaLeuLysLysGln 

226 TTGGGTGATAATGAAGCTATTACTCAAGAAATAGTGGGCTGTGCA 
LeuGlyAspAsnGluAlalleThrGlnGluIleValGlyCysAla 

2 71 CTTTGGAGAATTATGCTTTGAAAATGTTTTTGTATGCAGACAATG 
LeuTrpArglleMetLeu 

316 AAGATCGTGCTGGACGATTTCACAAAAACATGATCAAGTCCTTCT 
361 ATACTGCAAGTCTTTTGATAGATGTCATAACAGTATTTGGAGAAC 
4 06 TCACTGATGAAAATGTGAAACACAGGAAGTATGGCCAGAGGGAAG 
4 51 GCAACATACATCCATAATTGTTTNAAGANTGGGGAGACTCCCAAG 
496 CAGGCCCTGNTGGAATTGAAGA 



162 
SEQUENCE 18. IP-6 



1 GAGTATGAGATGAAACGAATGGCAGAGAATGAGCTGAGCCGGTCA 
GluTyrGluMetLysArgMetAlaGluAsnGluLeuSerArgSer 

4 6 GTAAATGAGTTTCTGTCCAAGCTGCAAGATGACCTCAAGGAGGCA 
ValAsnGluPheLeuSerLysLeuGlnAspAspLeuLysGluAla 

9 1 ATGAATACTATGATGTGTAGCCGATGCCAAGGAAAGCATAGGAGG 
MetAsnThrMetMetCysSerArgCysGlnGlyLysHisArgArg 

136 TTTGAAATGGACCGGGAACCTAAGAGTGCCAGATACTGTGCTGAG 
PheGluMetAspArgGluProLysSerAlaArgTyrCysAlaGlu 

181 TGTAATAGGCTGCATCCTGCTGAGGAAGGAGACTTTTGGGCAGAG 
CysAsnArgLeuHisProAlaGluGluGlyAspPheTrpAlaGlu 

226 TCAAGCATGTTGGGCCTCAAGATCACCTACTTTGCACTGATGGAT 
SerSerMetLeuGlyLeuLysIleThrTyrPheAlaLeuMetAsp 

2 71 GGAAAGGTGTATGACATCACAGAGTGGGCTGGATGCCAGCGTGTA 
GlyLysValTyrAspIleThrGluTrpAlaGlyCysGlnArgVal 

316 GGTATCTCCCCAGATACCCACAGAGTCCCCTATCACATCTCATTT 
GlylleSerProAspThrHisArgValProTyrHisIleSerPhe 

361 GGTTCTCGGATTCCAGGCACCAGAGGGCGGCAGAGAGCCACCCCA 
GlySerArglleProGlyThrArgGlyArgGlnArgAlaThrPro 

4 06 GATGCCCCTCCTGCTGATCTTCAGGATTTCTTGAGTCGGATCTTT 
AspAlaProProAlaAspLeuGlnAspPheLeuSerArgllePhe 

4 51 CAAGTACCCCCAGGGCAGATGCCAATGGGAACTTCTTTGCAGCTC 
GlnValProProGlyGlnMetProMetGlyThrSerLeuGlnLeu 

4 96 CTCAGCCTGCCCCTGGAGCCGCTGCAGCCTCTAAGCCCAACAGCA 
LeuSerLeuProLeuGluProLeuGlnProLeuSerProThrAla 

541 CAGTACCCAAGGGAGAAGCCAAACCTAAGCGGCGGAAGAAAGTGA 
GlnTyrProArgGluLysProAsnLeuSerGlyGlyArgLys 

586 GGAGGCCCTTCCAACGTTGATGCCCCTTCTCTTTCCTCAAATCAA 

631 TGTCAGGGAGTCAAAAGGGCTGTAGCACAGGATGGAGTTTGATTT 

6 76 ATCCCTCCTCCCCCAACACCTAGGAACTGAATCTTTTTCTTTTTA 

721 TTTTTTGAGATGGAGTCTTGCTCTGTTGCCCAG 



163 
SEQUENCE 19. IP-7 

I 

1 AAAACGGGTTTTCGCCATGTTGCTCAGGCTGGTCTTGAACTCCTG 

MetLeuLeuArgLeuValLeuAsnSerTr 

4 6 GGCTCAAGAATCTGTCTGCCTTAGCCAACCAAAGTGCTGGTATTA 
pAlaGlnGluSerValCysLeuSerGlnProLysCysTrpTyrTy 

9 1 CAGATGTGAGCTATTGCACGTGGCTAGGTTTCTCTGCTTTGAAGT 
rArgCysGluLeuLeuHisValAlaArgPheLeuCysPheGluVa 

136 TAATCTTTTTCTCCCTTTTCATAAATCTTTGGAAGGAAGTCACTG 
lAsnLeuPheLeuProPheHisLysSerLeuGluGlySerHisCy 

181 TGTGAAGCCTACACGTAGGAATGGGAAGTTACCCTGTTCCTCCAA 
sValLysProThrArgArgAsnGlyLysLeuProCysSerSerLy 

226 GGTGGTGGAGTATCTACATAATCTTCTGGAATTCTGTTCTGGAGA 
sValValGluTyrLeuHisAsnLeuLeuGluPheCysSerGlyGl 

2 71 GTTGCCTGTTCTCCCCCATTAATTAACTTATATCAGTATGGACNC 
uLeuProValLeuProHis 

316 ATGTATATTTACTTTATACCTTGGGTTAAAATCCAATACTAGTTA 
361 GATTTGTTGCTCAAATTGTNCCAGCTTTGGCCACTGAAACTCTTT 
4 06 CAGTTGGCAGTGCNGCTGTTAAAAANAAAAAAANAAAACCNGTT 



164 
SEQUENCE 20. IP-8 



I 

1 GGCCCCCTCGCTTCTGTGCAAGTATTTCCTGCAGCAGTGGGAACT 
AlaProSerLeuLeuCysLysTyrPheLeuGlnGlnTrpGluLe 

4 6 CACATCCCCTGGCCACGACACCTCGGTGCTGANTGACAGCGTGGA 
uThrSerProGlyHisAspThrSerValLeu AspSerValGl 

9 1 GATTGGCCTGCAGACCTGCTGCCACATCTTCCTCAACCTCGTGGT 
uIleGlyLeuGlnThrCysCysHisIlePheLeuAsnLeuValVa 

136 CACCGCACCGGGGCTGATCAAGCGTGACGCCTGCTTCACATCTCT 
lThrAlaProGlyLeuIleLysArgAspAlaCysPheThrSerLe 

181 AATGAACACCCTCATGACGTCGCTACCAGCACTAGTGCAGCAACA 
uMetAsnThrLeuMetThrSerLeuProAlaLeuValGlnGlnGl 

226 GGGAAGGCTGCTTCTGGCTGCTAATGTGGCCACCCTGGGGCTCCT 
nGlyArgLeuLeuLeuAlaAlaAsnValAlaThrLeuGlyLeuLe 

2 71 CATGGCCCGGCTCCTTAGCACCTCTCCAGCTCTTCAGGGAACACC 
uMetAlaArgLeuLeuSerThrSerProAlaLeuGlnGlyThrPr 

316 AGCATCCCGAGGGTTCTTCGCAGCTGCCATCCTCTTCCTATCACA 
oAlaSerArgGlyPhePheAlaAlaAlalleLeuPheLeuSerGl 

361 GTCCCACGTGGCGCGGGCCACCCCGGGCTCAGACCAGGCAGTGCT 
nSerHisValAlaArgAlaThrProGlySerAspGlnAlaValLe 

406 AGCCCTGTCCCCTGAGTATGAGGGCATCTGGGCCGACCTGCAGGA 
uAlaLeuSerProGluTyrGluGlylleTrpAlaAspLeuGlnGl 

451 GCTCTGGTTCCTGGGCATGCAGGCCTTCACCGGCTGTGTGCCTCT 
uLeuTrpPheLeuGlyMetGlnAlaPheThrGlyCysValProLe 

4 96 GCTGCCCTGGCTGGCCCCCGCTGCCCTG 
uLeuProTrpLeuAlaProAlaAla 



165 
SEQUENCE 21. IP-9 



1 GATGAGCATGGCAGTGCCAAGAACGTCAGCTTCAATCCTGCCAAG 
AspGluHisGlySerAlaLysAsnValSerPheAsnProAlaLys 

4 6 ATCAGTTCCAACTTCAGCAGCATTATTGCAGAGAAATTACGTTGT 
IleSerSerAsnPheSerSerllelleAlaGluLysLeuArgCys 

9 1 AATACCCTTCCTGACACTGGTCGCAGGAAGCCCCAAGTGAACCAG 
AsnThrLeuProAspThrGlyArgArgLysProGlnValAsnGln 

136 AAGGATAACTTCTGGCTGGTGACTGCACGATCCCAGAGTGCCATT 
LysAspAsnPheTrpLeuValThrAlaArgSerGlnSerAlalle 

181 AACACTTGGTTCACTGACTTGGCTGGCACCAAGCCACTCACGCAA 
AsnThrTrpPheThrAspLeuAlaGlyThrLysProLeuThrGln 

226 CTAGCCAAAAAGGTCCCCATTTTCAGTAAGAAGGAAGAGGTGTTT 
LeuAlaLysLysValProIlePheSerLysLysGluGluValPhe 

271 GGGTACTTAGCCAAATACACAGTGCCTGTGATGCGGGCTGCCTGG 
GlyTyrLeuAl aLysTyrThrVa 1 ProValMe t ArgAl aAl aTrp 

316 CTCATTAAGATGACCTGTGCCTACTATGCAGCAATCTCTGAGACC 
Leul 1 eLysMe tThr Cys Al aTyrTyr Al aAl a 1 1 eS erGluThr 

361 AAGGTTAAGAAGAGACATGTTGACCCTTTCATGGAATGGACTCAG 
LysValLysLysArgHisValAspProPheMetGluTrpThrGln 

406 ATCATCACCAAGTACTTATGGGAGCAGTTACAGAAGATGGCTGAA 
IlelleThrLysTyrLeuTrpGluGlnLeuGlnLysMetAlaGlu 

451 TACTACCGGCCAGGGCCTGCAGGAAGTGGGGGCTGTGGTTCCACG 
TyrTyrArgProGlyProAlaGlySerGlyGlyCysGlySerThr 

496 ATAGGGCCCTTGCCCCATGATGTAGAGGTGGCAATCCGGCAGTGG 
IleGlyProLeuProHisAspValGluValAlalleArgGlnTrp 

541 GATTACACCGAGAAGCTGGCCATGTTCATGTTTCAGGATGGAATG 
AspTyrThrGluLysLeuAlaMetPheMetPheGlnAspGlyMet 

586 CTGGACAGACATGAGTTCCTGACCTGGGTGCTTGAGTGTTTTGAG 
LeuAspArgHisGluPheLeuThrTrpValLeuGluCysPheGlu 

631 AAGATCCGCCCTGGAGAGGATGAATTGCTTAAACTGCTGCTGCCT 
LysIleArgProGlyGluAspGluLeuLeuLysLeuLeuLeuPro 

676 CTGCTTCTCCGATACTCTGGGGAATTTGTTCAGTCTGCATACCTG 
LeuLeuLeuArgTyrSerGlyGluPheValGlnSerAlaTyrLeu 

721 TCCCGCCGGCTTGCCTACTTCTGTACACGGAGACTGGCCCTGCAG 
SerArgArgLeuAlaTyrPheCysThrArgArgLeuAlaLeuGln 



166 



766 CTGGATGGTGTGAGCAGTCACTCATCTCATGTTATATCTGCTCAG 
LeuAspGlyValSerSerHisSerSerHisVallleSerAlaGln 

811 TCAACAAGCACGCTACCCACCACCCCTGCTCCTCAGCCCCCAACT 
SerThrSerThrLeuProThrThrProAlaProGlnProProThr 

856 AGCAGCACACCCTCGACTCCCTTTAGTGACCTGCTTATGTGCCCT 
SerSerThrProSerThrProPheSerAspLeuLeuMetCysPro 

901 CAGCACCGGCCCCTGGTTTTTGGCCTCAGCTGTATCCTACAGACC 
GlnHisArgProLeuValPheGlyLeuSerCysIleLeuGlnThr 

946 ATCCTCCTGTGCTGTCCTAGTGCCTTGGTTTGGCACTACTCACTG 
IleLeuLeuCysCysProSerAlaLeuValTrpHisTyrSerLeu 

991 ACTGATAGCAGAATTAAGACCGGCTCACCACTTGACCACTTGCCT 
ThrAspSerArglleLysThrGlySerProLeuAspHisLeuPro 

1036 ATTGCCCCGTCCAACCTGCCCATGCCAGAGGGTAACAGTGCCTTC 
IleAlaProSerAsnLeuProMetProGluGlyAsnSerAlaPhe 

1081 ACTCAGCAGGTCCGTGCAAAGTTGCGGGAGATCGAGCAGCAGATC 
ThrGlnGlnValArgAlaLysLeuArgGluIleGluGlnGlnlle 

1126 AAGGAGCGGGGACAGGCAGTTGAAGTTCGCTGGTCTTTCGATAAA 
LysGluArgGlyGlnAlaValGluValArgTrpSerPheAspLys 

1171 TGCCAGGAAGCTACTGCAGGCTTCACCATTGGACGGGTACTTCAT 
CysGlnGluAlaThrAlaGlyPheThrlleGlyArgValLeuHis 

1216 ACTTTGGAAGTGCTGGACAGCCATAGTTTTGAACGCTCTGACTTC 
ThrLeuGluValLeuAspSerHisSerPheGluArgSerAspPhe 

1261 AGCAACTCTCTTGACTCCCTTTGTAACCGAATCTTTGGATTGGGA 
SerAsnSerLeuAspSerLeuCysAsnArgllePheGlyLeuGly 

1306 CCTAGCAAGGATGGGCATGAGATCTCCTCAGATGATGATGCTGTG 
ProSerLysAspGlyHisGluIleSerSerAspAspAspAlaVal 

13 51 GTGTCATTGCTATGTGAATGGGCTGTCAGCTGCAAGCGTTCTGGT 
ValSerLeuLeuCysGluTrpAlaValSerCysLysArgSerGly 

1396 CGGCATCGTGCTATGGTGGTAGCCAAGCTCCTGGAGAAGAGACAG 
ArgHisArgAlaMetValValAlaLysLeuLeuGluLysArgGln 

1441 GCGGAGATTGAGGCTGAGCGTTGTGGAGAATCAGAAGCCGCAGAT 
AlaGluIleGluAlaGluArgCysGlyGluSerGluAlaAlaAsp 

1486 GAGAAGGGTTCCATCGCCTCTGGCTCCCTTTCTGCTCCCAGTGCT 
GluLysGlySerlleAlaSerGlySerLeuSerAlaProSerAla 

1531 CCCATTTTCCAGGATGTCCTCCTGCAGTTTCTGGATACACAGGCT 
ProIlePheGlriAspValLeuLeuGlnPheLeuAspThrGlnAla 



167 



1576 CCCATGCTGACGGACCCTCGAAGTGAGAGTGAGCGGGTGGAATTC 
ProMetLeuThrAspProArgSerGluSerGluArgValGluPhe 

1621 TTTAACTTAGTACTGCTGTTCTGTGAACTGATTCGACATGATGTT 
PheAsnLeuValLeuLeuPheCysGluLeuIleArgHisAspVal 

1666 TTCTCCCACAACATGTATACTTGCACTCTCATCTCCCGAGGGGAC 
PheSerHisAsnMetTyrThrCysThrLeuIleSerArgGlyAsp 

1711 CTTGCCTTTGGAGCCCCTGGTCCCCGGCCTCCCTCTCCCTTTGAT 
LeuAlaPheGlyAlaProGlyProArgProProSerProPheAsp 

1756 GATCCTGCCGATGACCCAGAGCACAAGGAGGCTGAAGGCAGCAGC 
AspProAlaAspAspProGluHisLysGluAlaGluGlySerSer 

18 01 AGCAGCAAGCTGGAAGATCCAGGGCTCTCAGAATCTATGGACATT 
SerSerLysLeuGluAspProGlyLeuSerGluSerMetAspIle 

1846 GACCCTAGTTCCAGTGTTCTCTTTGAGGACATGGAGAAGCCTGAT 
AspProSerSerSerValLeuPheGluAspMetGluLysProAsp 

18 91 TTCTCATTGTTCTCCCCTACTATGCCCTGTGAGGGGAAGGGCAGT 
PheSerLeuPheSerProThrMetProCysGluGlyLysGlySer 

1936 CCATCCCCTGAGAAGCCAGATGTCGAGAAGGAGGTGAAGCCCCCA 
ProSerProGluLysProAspValGluLysGluValLysProPro 

1981 CCCAAGGAGAAGATTGAAGGGACCCTTGGGGTTCTTTACGACCAG 
ProLysGluLysIleGluGlyThrLeuGlyValLeuTyrAspGln 

2 026 CCACGACACGTGCAGTACGCCACCCATTTTCCCATCCCCCAGGAG 
ProArgHisValGlnTyrAlaThrHisPheProIleProGlnGlu 

2 071 GAGTCATGCAGCCATGAGTGCAACCAGCGGTTGGTCGTACTGTTT 
GluSerCysSerHisGluCysAsnGlnArgLeuValValLeuPhe 

2116 GGGGTGGGAAAGCAGCGAGATGATGCCCGCCATGCCATCAAGAAA 
GlyValGlyLysGlnArgAspAspAlaArgHisAlalleLysLys 

2161 ATCACCAAGGATATCTTGAAGGTTCTGAACCGCAAAGGGACAGCA 
1 1 eThrLys Asp 1 1 eLeuLys Val LeuAsnArgLysGlyThr Ala 

2206 GAAACTGACCAGCTTGCTCCTATTGTGCCTCTGAATCCTGGAGAC 
GluThrAspGlnLeuAlaProIleValProLeuAsnProGlyAsp 

22 51 CTGACATTCTTAGGTGGGGAGGATGGGCAGAAGCGGCGACGCAAC 
LeuThrPheLeuGlyGlyGluAspGlyGlnLysArgArgArgAsn 

22 96 CGGCCTGAAGCCTTCCCCACTGCTGAAGATATCTTTGCTAAGTTC 
ArgProGluAlaPheProThrAlaGluAspIlePheAlaLysPhe 

2341 CAGCACCTTTCACATTATGACCAACACCAGGTCACGGCTCAGGTC 
GlnHisLeuSerHisTyrAspGlnHisGlnValThrAlaGlnVal 



168 



23 86 TCCCGGAATGTTCTGGAGCAGATCACGAGCTTTGCCCTTGGCATG 
SerArgAsnValLeuGluGlnlleThrSerPheAlaLeuGlyMet 

2431 TCATACCACTTGCCTCTGGTGCAGCATGTGCAGTTCATCTTCGAC 
SerTyrHisLeuProLeuValGlnHisValGlnPhellePheAsp 

2476 CTCATGGAATATTCACTCAGCATCAGTGGCCTCATCGACTTTGCC 
LeuMetGluTyrSerLeuSerlleSerGlyLeuIleAspPheAla 

2521 ATTCAGCTGCTGAATGAACTGAGTGTAGTTGAGGCTGAGCTGCTT 
IleGlnLeuLeuAsnGluLeuSerValValGluAlaGluLeuLeu 

2566 CTCAAATCCTCGGATCTGGTGGGCAGCTACACTACTAGCCTGTGC 
LeuLysSerSerAspLeuValGlySerTyrThrThrSerLeuCys 

2611 CTGTGCATCGTGGCTGTCCTGCGGCACTATCATGCCTGCCTCATC 
LeuCysIleValAlaValLeuArgHisTyrHisAlaCysLeuIle 

2656 CTCAACCAGGACCAGATGGCACAGGTCTTTGAGGGGCTGTGTGGC 
LeuAsnGlnAspGlnMetAlaGlnValPheGluGlyLeuCysGly 

2 701 GTCGTGAAGCATGGGATGAACCGGTCCGATGGCTCCTCTGCAGAG 
ValValLysHisGlyMetAsnArgSerAspGlySerSerAlaGlu 

2746 CGCTGTATCCTTGCTTATCTCTATGATCTGTACACCTCCTGTAGC 
ArgCysIleLeuAlaTyrLeuTyrAspLeuTyrThrSerCysSer 

2 791 CATTTAAAGAACAAATTTGGGGAGCTCTTCAGCGACTTTTGCTCA 
HisLeuLysAsnLysPheGlyGluLeuPheSerAspPheCysSer 

2836 AAGGTGAAGAACACCATCTACTGCAACGTGGAGCCATCGGAATCA 
LysValLysAsnThrlleTyrCysAsnValGluProSerGluSer 

2 881 AATATGCGCTGGGCACCTGAGTTCATGATCGACACTCTAGAGAAC 
AsnMetArgTrpAlaProGluPheMetlleAspThrLeuGluAsn 

2926 CCTGCAGCTCACACCTTCACCTACACGGGGCTAGGCAAGAGTCTT 
ProAlaAlaHisThrPheThrTyrThrGlyLeuGlyLysSerLeu 

2 971 AGTGAGAACCCTGCTAACCGCTACAGCTTTGTCTGCAATGCCCTT 

SerGluAsnProAlaAsnArgTyrSerPheValCysAsnAlaLeu 

3 016 ATGCACGTCTGTGTGGGGCACCATGATCCCGATAGGGTGAATGAC 

MetHisValCysValGlyHisHisAspProAspArgValAsnAsp 

3 061 ATCGCAATCCTGTGTGCAGAGCTGACCGGCTATTGCAAGTCACTG 
IleAlalleLeuCysAlaGluLeuThrGlyTyrCysLysSerLeu 

3106 AGTGCAGAATGGCTAGGAGTGCTTAAGGCCTTGTGCTGCTCCTCT 
SerAlaGluTrpLeuGlyValLeuLysAlaLeuCysCysSerSer 

3151 AACAATGGCACTTGTGGTTTCAACGATCTCCTCTGCAATGTTGAT 
AsnAsnGlyThrCysGlyPheAsnAspLeuLeuCysAsnValAsp 



169 



3196 GTCAGTGACCTATCTTTTCATGACTCGCTGGCTACTTTTGTTGCC 
ValSerAspLeuSerPheHisAspSerLeuAlaThrPheValAla 

3241 ATCCTCATCGCTCGGCAGTGTTTGCTCCTGGAAGATCTGATTCGC 
IleLeuIleAlaArgGlnCysLeuLeuLeuGluAspLeuIleArg 

3 2 8 S TGTGCTGCCATCCCTTCACTCCTTAATGCTGCTTGTAGTGAACAG 
CysAlaAlalleProSerLeuLeuAsnAlaAlaCysSerGluGln 

3 331 GACTCTGAGCCAGGGGCCCGGCTTACCTGCCGCATCCTCCTTCAC 
AspSerGluProGlyAlaArgLeuThrCysArglleLeuLeuHis 

33 76 CTTTTCAAGACACCGCAGCTCAATCCTTGCCAGTCTGATGGAAAC 
LeuPheLysThrProGlnLeuAsnProCysGlnSerAspGlyAsn 

3421 AAGCCTACAGTAGGAATCCGCTCCTCCTGCGACCGCCACCTGCTG 
LysProThrValGlylleArgSerSerCysAspArgHisLeuLeu 

3466 GCTGCCTCCCAGAACCGCATCGTGGATGGAGCCGTGTTTGCTGTT 
AlaAlaSerGlnAsnArglleValAspGlyAlaValPheAlaVal 

3 511 CTCAAGGCTGTGTTTGTACTTGGGGATGCGGAACTGAAAGGTTCA 
LeuLysAlaValPheValLeuGlyAspAlaGluLeuLysGlySer 

3556 GGCTTCACTGTGACAGGAGGAACAGAAGAACTTCCAGAGGAGGAG 
GlyPheThrValThrGlyGlyThrGluGluLeuProGluGluGlu 

3601 GGAGGAGGTGGCAGTGGTGGTCGGAGGCAGGGTGGCCGCAACATC 
GlyGlyGlyGlySerGlyGlyArgArgGlnGlyGlyArgAsnlle 

3646 TCTGTGGAGACAGCCAGTCTGGATGTCTATGCCAAGTACGTGCTG 
SerValGluThrAlaSerLeuAspValTyrAlaLysTyrValLeu 

36 91 CGCAGCATCTGCCAACAGGAATGGGTAGGAGAACGTTGCCTTAAG 
ArgSerlleCysGlnGlnGluTrpValGlyGluArgCysLeuLys 

3 736 TCTCTGTGTGAGGACAGCAATGACCTGCAAGACCCAGTGTTGAGT 
SerLeuCysGluAspSerAsnAspLeuGlnAspProValLeuSer 

3 781 AGTGCCCAGGCGCAGCGCCTCATGCAGCTCATTTGCTATCCACAT 
SerAlaGlnAlaGlnArgLeuMetGlnLeuIleCysTyrProHis 

3826 CGACTGCTGGACAATGAGGATGGGGAAAACCCCCAGCGGCAGCGC 
ArgLeuLeuAspAsnGluAspGlyGluAsnProGlnArgGlnArg 

3 8 71 ATAAAGCGCATTCTCCAGAACTTGGACCAGTGGACCATGCGCCAG 
I 1 eLys Arg I 1 eLeuGlnAsnLeuAspGlnTrpThrMe t ArgGln 

3 916 TCTTCCTTGGAGCTGCAGCTCATGATCAAGCAGACCCCTAACAAT 
SerSerLeuGluLeuGlnLeuMetlleLysGlnThrProAsnAsn 

3 961 GAGATGAACTCCCTCTTGGAGAACATCGCCAAGGCCACAATCGAG 
GluMetAsnSerLeuLeuGluAsnlleAlaLysAlaThrlleGlu 



170 



4 006 GTTTTCCAACGGTCAGCAGAGACAGGGTCATCTTCTGGAAGTACT 
ValPheGlnArgSerAlaGluThrGlySerSerSerGlySerThr 

4 051 GCAAGCAACATGCCCAGCAGCAGCAAGACCAAGCCTGTGCTCAGC 
AlaSerAsnMetProSerSerSerLysThrLysProValLeuSer 

4 096 TCTCTAGAGCGCTCTGGTGTATGGCTGGTGGCCCCCCTCATTGCT 
SerLeuGluArgSerGlyValTrpLeuValAlaProLeuIleAla 

4141 AAACTGCCCACCTCAGTCCAGGGACATGTGTTAAAGGCTGCTGGG 
LysLeuProThrSerValGlnGlyHisValLeuLysAlaAlaGly 

4186 GAAGAATTGGAGAAGGGTCAGCACCTGGGTTCCTCTTCACGCAAA 
GluGluLeuGluLysGlyGlnHisLeuGlySerSerSerArgLys 

4231 GAACGTGATCGACAAAAGCAGAAGAGCATGTCCCTATTGAGCCAG 
GluArgAspArgGlnLysGlnLysSerMetSerLeuLeuSerGln 

4276 CAGCCCTTCTTATCGCTGGTGCTAACATGTCTGAAAGGGCAGGAT 
GlnProPheLeuSerLeuValLeuThrCysLeuLysGlyGlnAsp 

4321 GAACAACGCGAGGGACTCCTTACCTCCCTCTACAGCCAGGTGCAC 
GluGlnArgGluGlyLeuLeuThrSerLeuTyrSerGlnValHis 

4366 CAGATTGTGAATAATTGGCGAGATGACCAGTACTTAGATGATTGC 
GlnlleValAsnAsnTrpArgAspAspGlnTyrLeuAspAspCys 

4411 AAACCAAAGCAGCTTATGCATGAGGCACTCAAACTGCGGCTCAAC 
LysProLysGlnLeuMetHisGluAlaLeuLysLeuArgLeuAsn 

4456 CTGGTGGGGGGCATGTTTGACACGGTGCAGCGCAGCACCCAGCAG 
LeuValGlyGlyMetPheAspThrValGlnArgSerThrGlnGln 

4 501 ACCACGGAGTGGGCCATGCTCCTCCTGGAGATCATCATCAGCGGC 
ThrThrGluTrpAlaMetLeuLeuLeuGluIlellelleSerGly 

4546 ACTGTCGACATGCAGTCCAACAATGAGCTCTTCACTACTGTGTTG 
ThrValAspMetGlnSerAsnAsnGluLeuPheThrThrValLeu 

4591 GACATGCTGAGCGTGCTCATCAATGGGACATTGGCTGCAGACATG 
AspMetLeuSerValLeuIleAsnGlyThrLeuAlaAlaAspMet 

4636 TCTAGCATCTCGCAAGGTAGCATGGAGGAAAACAAGCGTGCATAC 
SerSerlleSerGlnGlySerMetGluGluAsnLysArgAlaTyr 

4681 ATGAACCTGGCGAAGAAGTTGCAGAAGGAGTTGGGGGAGCGCCAG 
MetAsnLeuAlaLysLysLeuGlnLysGluLeuGlyGluArgGln 

4726 TCAGACAGTCTGGAAAAGGTTCGCCAGCTGCTGCCACTGCCCAAG 
SerAspSerLeuGluLysValArgGlnLeuLeuProLeuProLys 

4 771 CAGACCCGAGATGTCATCACGTGTGAGCCACAGGGCTCCCTTATC 
GlnThrArgAspVallleThrCysGluProGlnGlySerLeuIle 



171 



4816 GATACCAAGGGCAACAAGATTGCTGGCTTCGATTCCATCTTCAAG 
AspThrLysGlyAsnLysIleAlaGlyPheAspSerllePheLys 

4861 AAGGAGGGTCTACAGGTTTCCACCAAACAGAAGATCTCGCCCTGG 
LysGluGlyLeuGlnValSerThrLysGlnLysIleSerProTrp 

4 906 GATCTTTTTGAGGGGTTGAAGCCGTCAGCACCACTCTCTTGGGGC 
AspLeuPheGluGlyLeuLysProSerAlaProLeuSerTrpGly 

4 951 TGGTTTGGAACAGTCCGAGTGGACCGGCGAGTGGCTCGAGGAGAG 
TrpPheGlyThrValArgValAspArgArgValAlaArgGlyGlu 

4 996 GAGCAGCAGCGGTTGCTGCTCTACCACACACACCTGAGGCCCCGG 
GluGlnGlnArgLeuLeuLeuTyrHisThrHisLeuArgProArg 

5041 CCCCGCGCCTATTACCTGGAGCCACTGCCACTGCCCCCAGAAGAT 
ProArgAlaTyrTyrLeuGluProLeuProLeuProProGluAsp 

5086 GAGGAGCCGCCTGCTCCTACCCTGCTAGAGCCTGAGAAAAAGGCT 
GluGluProProAlaProThrLeuLeuGluProGluLysLysAla 

5131 CCAGAGCCCCCCAAAACTGACAAACCGGGGGCTGCTCCACCCAGT 
ProGluProProLysThrAspLysProGlyAlaAlaProProSer 

5176 ACTGAGGAACGCAAGAAGAAGTCCACCAAGGGCAAGAAACGCAGC 
ThrGluGluArgLysLysLysSerThrLysGlyLysLysArgSer 

5221 CAGCCAGCTACCAAGACAGAGGACTATGGAATGGGCCCGGGTCGG 
GlnProAlaThrLysThrGluAspTyrGlyMetGlyProGlyArg 

5266 AGCGGCCCTTATGGTGTGACAGTGCCTCCGGACCTCCTGCACCAC 
SerGlyProTyrGlyValThrValProProAspLeuLeuHisHis 

5311 CCAAACCCTGGTTCTATAACACACCTTAACTACAGGCAAGGCTCC 
ProAsnProGlySerlleThrHisLeuAsnTyrArgGlnGlySer 

5356 ATAGGCCTGTACACCCAGAACCAGCCACTACCTGCAGGTGGCCCT 
IleGlyLeuTyrThrGlnAsnGlnProLeuProAlaGlyGlyPro 

54 01 CGTGTGGACCCATACCGTCCTGTGCGCTTACCAATGCAGAAGCTG 
ArgValAspProTyrArgProValArgLeuProMetGlnLysLeu 

5446 CCCACCCGACCAACTTACCCTGGAGTGCTGCCCACAACCATGACT 
ProThrArgProThrTyrProGlyValLeuProThrThrMetThr 

54 91 GGCGTCATGGGTTTAGAACCCTCCTCTTATAAGACCTCTGTGTAC 
GlyValMetGlyLeuGluProSerSerTyrLysThrSerValTyr 

5536 CGGCAGCAGCAACCTGCGGTGCCCCAAGGACAGCGCCTTCGCCAA 
ArgGlnGlnGlnProAlaValProGlnGlyGlnArgLeuArgGln 

5581 CAGCTCCAGCAGAGTCAGGGCATGTTGGGACAGTCATCTGTCCAT 
GlnLeuGlnGlnSerGlnGlyMetLeuGlyGlnSerSerValHis 



172 



5626 CAGATGACTCCCAGCTCTTCCTACGGTTTGCAGACTTCCCAGGGC 
GlnMetThrProSerSerSerTyrGlyLeuGlnThrSerGlnGly 

5671 TATACTCCTTATGTTTCTCATGTGGGATTGCAGCAACACACAGGC 
TyrThrProTyrValSerHisValGlyLeuGlnGlnHisThrGly 

5716 CCTGCAGGTACCATGGTGCCCCCCAGCTACTCCAGCCAGCCTTAC 
ProAlaGlyThrMetValProProSerTyrSerSerGlnProTyr 

5761 CAGAGCACCCACCCTTCTACCAATCCTACTCTTGTAGATCCTACC 
GlnSerThrHisProSerThrAsnProThrLeuValAspProThr 

58 06 CGCCACCTGCAACAGCGGCCCAGTGGCTATGTGCACCAGCAGGCC 
ArgHisLeuGlnGlnArgProSerGlyTyrValHisGlnGlnAla 

5851 CCCACCTATGGACATGGACTGACCTCCACTCAAAGGTTTTCACAC 
ProThrTyrGlyHisGlyLeuThrSerThrGlnArgPheSerHis 

5896 CAGACACTGCAGCAGACACCCATGATAAGTACCATGACTCCAATG 
GlnThrLeuGlnGlnThrProMetlleSerThrMetThrProMet 

5 941 AGTGCCCAGGGCGTCCAGGCAGGCGTCCGTTCAACAGCCATCCTA 

SerAlaGlnGlyValGlnAlaGlyValArgSerThrAlalleLeu 

5986 CCTGAGCAGCAGCAGCAGCAGCAACAGCAGCAACAGCAACAGCAG 
ProGluGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGln 

6 031 CAGCAGCAGCAACAGCAACAGCAGCAGCAGCAGCAGCAGTACCAC 

GlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnTyrHis 

6076 ATCCGGCAGCAGCAGCAGCAGCAGATCCTGCGGCAGCAGCAGCAA 
IleArgGlnGlnGlnGlnGlnGlnlleLeuArgGlnGlnGlnGln 

6121 CAGCAACAGCAGCAGCAGCAGCAGCAGCAACAGCAACAGCAGCAG 
GlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGlnGln 

6166 CAGCAACAGCAACAACAGCAA.CACCAGCAGCAACAGCAGCAACAG 
GlnGlnGlnGlnGlnGlnGlnHisGlnGlnGlnGlnGlnGlnGln 

6211 GCGGCTCCTCCCCAACCCCAGCCCCAGTCCCAGCCCCAGTTCCAG 
AlaAlaProProGlnProGlnProGlnSerGlnProGlnPheGln 

62 56 CGCCAGGGGCTTCAGCAGACCCAGCAGCAGCAACAGACAGCAGCT 

ArgGlnGlyLeuGlnGlnThrGlnGlnGlnGlnGlnThrAlaAla 

6301 TTGGTCCGGCAACTTCAACAACAGCTCTCTAATACCCAGCCACAG 
LeuValArgGlnLeuGlnGlnGlnLeuSerAsnThrGlnProGln 

6346 CCCAGTACCAACATATTTGGACGCTACTGAGCCACCTGGAGGAAC 
ProSerThrAsnllePheGlyArgTyr 

63 91 TGCTTGTGCACTGGATGTGGCCCCACCCTTTCCTCTTAATTCCCA 

64 36 ATCCCATTCCTGGGCTAGCACCAGTAGTGGTTGGGGCCCTCCCCT 
6481 CAGGCTCCATTTTTAATAAGTTTTTAGTATTTTTGTTAATGTGAG 
6526 GCATTGAGCTGTTGGGTTTTGTATATTATTTATATAGAGACCCCA 
6571 GAGCTGTTGCACCCAATACACAGAGCTTCTTTGC 



173 
SEQUENCE 22. IP-10 



I 



1 GTTTAAAGCTGAGAACGCATCTTTAGCTAAACTTCGCATTGAACG 
PheLysAlaGluAsnAlaSerLeuAlaLysLeuArglleGluAr 

4 6 AGAAAGTGCCTTGGAAAAACTCAGGAAAGAAATTGCAGACTTCGA 
gGluSerAlaLeuGluLysLeuArgLysGluIleAlaAspPheGl 

9 1 ACAACAGAAAGCAAAAGAATTAGCTCGAATAGAAGAGTTTAAAAA 
uGlnGlnLysAlaLysGluLeuAlaArglleGluGluPheLysLy 

136 GGAGGAGATGAGGAAGCTACAAAAGGAACGTAAAGTTTTTGAAAA 
sGluGluMetArgLysLeuGlnLysGluArgLysValPheGluLy 

181 GTATACTACAGCTGCAAGAACTTTTCCAGATAAAAAGGAACGTGA 
sTyrThrThrAlaAlaArgThrPheProAspLysLysGluArgGl 

226 AGAAATACAGACTTTAAAACAGCAAATAGCAGATTTACGGGAAGA 
uGluIleGlnThrLeuLysGlnGlnlleAlaAspLeuArgGluAs 

271 TTTGAAAAGAAAGGAGACCAAATGGTCAAGTACACACAGCCGTCT 
pLeuLysArgLysGluThrLysTrpSerSerThrHisSerArgLe 

316 CAGAAGCCAGATACAAATGTTAGTCAGAGAGAACACAGACCTCCG 
uArgSerGlnlleGlnMetLeuValArgGluAsnThrAspLeuAr 

361 GGAAGAAATAAAAGTGATGGAAAGATTCCGACTGGATGTCTGGAA 
gGluGluIleLysValMetGluArgPheArgLeuAspValTrpLy 

4 06 GAGAGCAGAAGCCATAGAGAGCAGCCTCGAGGTGGAGGAGGAGGG 
sArgAlaGluAlalleGluSerSerLeuGluValGluGluGluGl 

451 C 

y 



174 
SEQUENCE 23. IP-ll 



1 AATTCGCGGNGGCTCGACGCCGCGCCACCGGCCCGGCAGGTGCTG 
PheAla AlaArgArgArgAlaThrGlyProAlaGlyAlaV 

4 6 TCCTTATTCCCAGCCCAGTCAAGAGCTACCGGGGCTGGCTAGTCA 
alLeuIleProSerProValLysSerTyrArgGlyTrpLeuValM 

9 1 TGGGGGAGCCCAGTAGAGAGGAGTATAAAATCCAGTCCTTTGATG 
etGlyGluProSerArgGluGluTyrLysIleGlnSerPheAspA 

136 CAGAGACCCAGCAGCTGCTGAAGACAGCACTCAAAGATCCGGGTG 
laGluThrGlnGlnLeuLeuLysThrAlaLeuLysAspProGlyA 

181 CTGTGGACTTGGAGAAAGTGGCCAATGTGATTGTGGACCATTCTC 
laValAspLeuGluLysValAlaAsnVallleValAspHisSerL 

226 TGCAGGACTGTGTGTTCAGCAAGGAAGCAGGACGCATGTGCTACG 
euGlnAspCysValPheSerLysGluAlaGlyArgMetCysTyrA 

271 CCATCATTCAGGCAGAGAGTAAACAAGCAGGCCAGAGTGTCTTCC 
lallelleGlnAlaGluSerLysGlnAlaGlyGlnSerValPheA 

316 GACGTGGACTCCTCAACCGGCTGCAGCAGGAGTACCAGGCTCGGG 
rgArgGlyLeuLeuAsnArgLeuGlnGlnGluTyrGlnAlaArgG 

361 AGCAGCTGCGAGCACGCTCCCTGCAGGGCTGGGTCTGCTATGTCA 
luGlnLeuArgAlaArgSerLeuGlnGlyTrpValCysTyrValT 

406 CCTTTATCTGCAACATCTTTGACTACCTGAGGGTGAACAACATGC 
hrPhelleCysAsnllePheAspTyrLeuArgValAsnAsnMetP 

451 CCATGATGGCCCTGGTGAACCCTGTCTATGACTGCCTCTTCCGGC 
roMetMetAlaLeuValAsnProValTyrAspCysLeuPheArgL 

4 96 TT 

eu 



175 
SEQUENCE 24. IP-12 



1 CTTCTTCTCTCTTTGCTTCAGACTGGCCCGGGAGCAAGCGCGAGT 
PhePheSerLeuCysPheArgLeuAlaArgGluGlnAlaArgVa 

4 6 GTGCGAACTGCAGAGTGGGAACCAGCAGCTGGAGGAGCAGCGGGT 
ICysGluLeuGlnSerGlyAsnGlnGlnLeuGluGluGlnArgVa 

9 1 GGAGCTGGTGGAAAGACTGCAGGCCATGCTGCAGGCCCACTGGGA 
lGluLeuValGluArgLeuGlnAlaMetLeuGlnAlaHisTrpAs 

136 TGAGGTCAACCAGCTGCTCAGCACCACTCTCCCGCCGCCCAACCC 
pGluValAsnGlnLeuLeuSerThrThrLeuProProProAsnPr 

181 TCAAGCTCCTCCTGATGGACCCTCCAGCCCCGGGCCTCAGGAGAC 
oGlnAlaProProAspGlyProSerSerProGlyProGlnGluTh 

226 GGAGAAGGAGGAGAGGAGGGTTTGGACTATCCCTCCCATGGCCGT 
rGluLysGluGluArgArgValTrpThrlleProProMetAlaVa 

271 GGCCTCAACCCTGTATTCCAACAGAGCCGGGAAGCAAGGCACGAG 
lAlaSerThrLeuTyrSerAsnArgAlaGlyLysGlnGlyThrSe 

316 TTACCTGGACCGCCTCCTGTTCTTTTCAATTTCTTCTCAGATCTT 
rTyrLeuAspArgLeuLeuPhePheSerlleSerSerGlnlleLe 

361 AGCCTCCTGTTGGGCCCCTCTTTTCATAATCAACATTTTTTC 
uAlaSerCysTrpAlaProLeuPhellelleAsnllePhe 



176 
SEQUENCE 25. IP-13 



1 GCGAGCGTGAGTCAGCGCTGAAGGCCCAGAGCGCGCTGCACGAGC 
GluArgGluSerAlaLeuLysAlaGlnSerAlaLeuHisGluG 



GAI 



4 6 AGAAGACTCTGCCCGGGATGAACCGGCCGATCCAGGTGAAGCTGC 
InLysThrLeuProGlyMetAsnArgProIleGlnValLysLeuA 

9 1 GAACACGAGACCGAGGAGATAGAAAACTCTTCGTGGGCATGCTCA 
rgThrArgAspArgGlyAspArgLysLeuPheValGlyMetLeuA 

136 ACAAGCAAAAGTCCGAGGACGACGTGCGCCGCCTTTTCGAGGCCT 
snLysGlnLysSerGluAspAspValArgArgLeuPheGluAlaP 

181 TTGGGAACATCGAGGAGTGCACCATCCTGCGCGGGCCCGACGGCA 
heGlyAsnlleGluGluCysThrlleLeuArgGlyProAspGlyA 

226 ACAGCAAGGGGTGCGCCTTTGTGAAGTACTCCTCCCACGCCGAGG 
snSerLysGlyCysAlaPheValLysTyrSerSerHisAlaGluG 

271 GGCAGGCCGCCATTCAACGCGCTACACGGCAGCCAGACCATGCCG 
lyGlnAlaAlalleGlnArgAlaThrArgGlnProAspHisAlaG 

316 GGAGCCTCGTCCAGTCTGGTGGTCAAGTTCGCCGACACCGACAAG 
lySerLeuValGlnSerGlyGlyGlnValArgArgHisArgGlnG 

361 GAGCGCACGATGCGGCGAATGCAGCAGATGGTTGGCCAGATGGGC 
lyAlaHisAspAlaAlaAsnAlaAlaAspGlyTrpProAspGlyH 

406 ATGTTCAACCCCATGGCCATCCCTTTCGGGGCCTACGGGCCTACG 
isValGlnProHisGlyHisProPheArgGlyLeuArgAlaTyrA 

451 CTCAGGCAATGCAGCAGCAAGCGGCCCTGATGGCATCAGTCGCGC 
laGlnAlaMetGlnGlnGlnAlaAlaLeuMetAlaSerValAlaG 

496 AGGGGGCCTACCTGAACCCCATGGCTGCCTTCGCTGTCGCCCAGA 
InGlyAlaTyrLeuAsnProMetAlaAlaPheAlaValAlaGlnM 

541 TGCAGCAGATGGCG 
etGlnGlnMetAla 



BIBLIOGRAPHY 



Aberanthy CR, Rasmussen SA, Stalker HJ, Zori R, Driscoll DJ, Williams CA, Kousseff 
BG, Wallace MR. (1997) NF1 mutation analysis using a combined heteroduplex/SSCP 
approach. Hum Mutation 9: 548-554. 

Adams MD, Kerlavage AR, Fleischmann RD, Fuldner RA, Bult CJ, Lee NH, Kirkness 
EF, Weinstock KG, Gocayne JD, White O. (1995) Initial assessment of human gene 
diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. 
Nature 311 (6547 Suppl): 3-174. 

Adams MD, Soares MB, Kerlavage AR, Fields C, Venter JC. (1993) Rapid cDNA 
sequencing (expressed sequence tags) from a directionally cloned human infant brain 
cDNA library. Nature Genetics 4 (4): 373-380. 

Aitken A, Collinge DB, van Heusden BP, Isobe T, Roseboom PH, Rosenfeld G, Soil J. 
(1992) 14-3-3 proteins: a highly conserved, widespread family of eukaryotic proteins. 
Trends Biochem Sci 17(12): 498-501. 

Aitken A, Jones D, Soneji Y, Howell S. (1995) 14-3-3 proteins: biological function and 
domain structure. Biochemical Society Transactions 23(3): 605-1 1. 

Alberts B, Bray D, Lewis J, Raff, M, Roberts K, Watson JD. (1994) Molecular biology of 
the cell. Garland Publishing, Inc., New York, NY. 

Allende JE, Allende CC. (1995) Protein kinase CK2: an enzyme with multiple substrates 
and a puzzling regulation. FASEBJ9: 313-23. 

Altschul SF, Gish W, Miller H, Myers EW, Lipman DJ. (1990) Basic local alignment 
search tool. J Mol Biol, 215: 403-410. 

Apitz-Castro R, Cruz MR, Ledezma E, Merino F, Ramirez-Duque P, Dangelmeier C, 
Holmsen H. (1985) The storage pool deficiency in platelets from humans with the 
Chediak-Higashi syndrome: study of six patients. Br J Hematol 59(3): 471-83. 

Arden SD, Roep BO, Neophytou PI, Usac EF, Duinkerken G, de Vries RRP, Hutton JC. 
(1996) Imogen 38: a novel 38-kD islet mitochondrial autoantigen recognized by T cells 
from a newly diagnosed type 1 diabetic patient. J Clin Invest 97: 551-61. 



177 



178 



Asao H, Sasaki Y, Arita T, Tanaka N, Endo K, Kasai H, Takeshita T, Endo Y, Fujita T, 
Sugamura K. (1997) Hrs is associated with STAM, a signal transducing adaptor 
molecule. J Biol Chem 272: 32785-91. 

Baetz K, Isaaz S, Griffiths GM. (1995) Loss of cytotoxic T lymphocyte function in 
Chediak-Higashi syndrome arises from a secretory defect that prevents lytic granule 
exocytosis. J Immunol 154: 6122-31. 

Bairoch A. (1992) PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids 
Research 20 Suppl:20 13-20 18. 

Ballard R, Tien RD, Nohria V, Juel V. (1994) The Chediak-Higashi syndrome: CT and 
MR findings. Pediatr Radiol 24(4), 266-7. 

Barbosa MDFS, Barrat FJ, Tchernev VT, Nguyen QA, Mishra VS, Colman SD, Pastural 
E, Dufourcq-Lagelouse R, Fischer A, Holcombe RF, Wallace MR, Brandt SJ, de Saint 
Basile G, Kingsmore SF. (1997). Identification of mutations in two major mRNA 
isoforms of the Chediak-Higashi syndrome gene in human and mouse. Hum Mol Genet 
6,1091-1098. 

Barbosa MDFS, Nguyen QA, Tchernev VT, Ashley JA, Detter CD, Blaydes SM, Brandt 
SJ, Chotai D, Hodgman C, Solari RCE, Lovett M, Kingsmore, SF. (1996) Identification 
of the homologous beige and Chediak-Higashi syndrome genes. Nature 382:262-5. 

Barrat FJ, Auloge L, Pastural E, Dufourcq-Lagelouse R, Vilmer E, Cant AJ, Weissenbach 
J, Le Paslier D, FischerA, de Saint Basile G. (1996) Genetic and physical mapping of the 
Chediak-Higashi syndrome on Chromosome lq42-43. Am J Hum Genet 59:625-32. 

Bean AJ, Seifert R, Chen YA, Sacks R, Scheller RH. (1997) Hrs-2 is an ATPase 
implicated in calcium-regulated sectretion. Nature385: 826-29. 

Beguez-Cesar A. (1943) Neutropenia cronica maligna familiar con granulaciones atipicas 
de los leucocitos. Bol Soc Cubana Pediat. 15, 900-922. 

Beidler JL, Hilliard PR, Rill RL. (1982) Ultrasensitive staining of nucleic acids with 
silver. Anal Biochem 126: 374-80. 

Bellinati-Pires R, Salgado MM, Joazerio PP, Carneiro-Sampaio MM. (1992) Delayed 
phagocytosis and bacterial killing in Chediak-Higashi syndrome neutrophils detected by a 
fluorochrome assay. Mem Inst Oswaldo Cruz 87(4):575-81. 

Belmont LD, Mitchison TJ. (1996) Identification of a protein that interacts with tubulin 
dimers and increases the catastrophe rate of microtubules. Cell 84:623-31. 



179 



Benfenati F, Valtorta F, Chieregatti E, Greengard P. (1992) Interaction of free and 
synaptic vesicle-bound synapsin I with F-actin. Neuron 8: 377-86. 

Bennet MK, Miller KG, Scheller RH. (1993) Casein kinase II phosphorylates the synaptic 
vesicle protein p65. J Neuroscience 13: 1701-07. 

Bishop DT. (1985) The information content of phase-known matings for ordering genetic 
loci. Genet Epidemiol, 2, 349-361. 

Blume RS, Bennett JM, Yankee RA, Wolff SM. (1968) Defective granulocyte regulation 
in the Chediak-Higashi syndrome. N EnglJ Med, 279(19): 1009-15. 

Blume RS, Wolff SM. (1972). The Chediak-Higashi syndrome: studies in four patients 
and a review of the literature. Medicine Baltimore 51(4), 247-80. 

Brahmi Z. (1983) Nature of natural killer cell hyporesponsiveness in the Chediak-Higashi 
syndrome. Hum Immunol 6(1): 45-52. 

Breeden L, Nasmyth K. (1985) Regulation of the yeast HO gene. Cold Spring Harbor 
Quant Biol 50: 643-650. 

Burbelo PD, Hall A. (1995) Hot numbers in signal transduction. Current Biology 5(2): 
95-6. 

Burgoyne RD, Cheek TR, Norman KM. (1986) Identification of a secretory granule- 
binding protein as caldesmon. Nature 319: 68-70. 

Burgoyne RD, Morgan A, Robinson I, Pender N, Cheek T. (1993) Exocytosis in adrenal 
chromaffin cells. JAnat 183:309-14. 

Burkhardt JK, Wiebel FA, Hester S, Argon Y. (1993) The giant organelles in beige and 
Chediak-Higashi fibroblasts are derived from late endosomes and mature lysosomes. J 
Exp Med. 178(6): 1845-56. 

Cairo MS, Vandeven C, Toy C, Tischler D, Sender L. (1988) Fluorescent cytometric 
analysis of polymorphonuclear leukocytes in Chediak-Higashi syndrome: diminished 
C3bi receptor expression (OKM1) with normal granular cell density. Pediatr Res 
24(6):673-6. 

Chamberlain LH, Roth D, Morgan A, Burgoyne RD. (1995) Distinct effects of a-SNAP, 
14-3-3 proteins and calmodulin on priming and triggering of regulated exocytosis. J Cell 
Biol 130: 1063-70. 



180 



Chediak M. (1952) Nouvelle anomalie leucocytaire de caractere constitutionnel et 
familial. Rev Hematol 7, 362. 

Church GM, Gilbert W. (1984) Genomic sequencing. Proc Natl Acad Sci USA, 81, 1991- 
1995. 

Clark EA, Roths JB, Murphy ED, Ledbetter JA, Clagett JA. (1982) The beige (bg) gene 
influences the development of autoimmune disease in SB/Le male mice. In: NK cells and 
other natural effector cells. Heberman-RB (ed). Academic Press, p. 301-306. 

Clawson CC, White JG, Repine JE. (1978) The Chediak-Higashi syndrome. Evidence 
that defective leukotaxis is primarily due to an impediment by giant granules. Am J 
Pathol 92(3): 745-53. 

Colgan SP, Blancquaert AM, Thrall MA, Bruyninckx WJ. (1992) Defective in vitro 
motility of polymorphonuclear leukocytes of homozygote and heterozygote Chediak- 
Higashi cats. Vet Immunol Immunopoathol 3 1(3-4): 205-27. 

Colgan SP, Gasper PW, Thrall MA, Boone TC, Blancquaert AM, Bruyninckx WJ. (1992) 
Neutrophil function in normal and Chediak-Higashi syndrome cats following 
administration of recombinant canine granulocyte colony-stimulating factor. Exp 
Hematol 20(10): 1229-34. 

Colgan SP, Hull-Thrall MA, Gasper PW. (1989) Platelet aggregation and ATP secretion 
in whole blood of normal cats and cats homozygous and heterozygous for Chediak- 
Higashi syndrome. Blood Cells 15(3): 585-95. 

Davletov B, Sontag JM, Hata Y, Petrenko AG, Fyks EM, Jahn R, Suedhof TC. (1993) 
Phosphorylation of synaptotagmin I by casein kinase II. J Biol Chem 268:6816-22. 

DeLorenzo RJ. (1981) The calmodulin hypothesis of neurotransmission. Cell Calcium 2: 
365-85. 

Devereux J, Haeberli M, Smithies O. (1984) A comprehensive set of sequence analysis 
programs for the VAX. Nucleic Acids Res, 12, 387-395. 

Fu H, Xia K, Pallas DC, Cui C, Conroy K, Narsimhan RP, Mamon H, Collier RJ, Roberts 
TM. (1994) Interaction of the protein kinase Raf-1 with 14-3-3 proteins. Science 266: 
126-29. 

Fujita Y, Fukui K, Kotani H, Kimura T, Hata Y, Sudhof TC, Scheller RH, Takai Y. 
(1996) J Biol Chem 271: 7265-68. 

Fukai K, Oh J, Karim MA, Moore KJ, Kandil HH, Ito H, Buerger J, Spritz RA. (1996) 
Homozygosity mapping of the gene for Chediak-Higashi syndrome to chromosome lq42- 



181 



q44 in a segment of conserved synteny that includes the mouse beige locus (bg). Am J 
Hum Genet 59:620-624. 

Gallin JI, Bujak JS, Patten E, Wolff SM. (1974) Granulocyte function in the 
Chediak-Higashi syndrome of mice. Blood 43(2): 201-6. 

Gallin JI, Klimerman JA, Padgett GA, Wolff SM. (1975) Defective mononuclear 
leukocyte chemotaxis in the Chediak-Higashi syndrome of humans, mink, and cattle. 
Blood 45(6): 863-70. 

Gish W, States DJ. (1993) Identification of protein coding regions by database similarity 
search. Nat Genet 3(3):266-272. 

Gong W, Emanuel BS, Collins J, Kim DH, Wang Z, Chen F, Zhang G, Roe B, Budarf M. 
(1996) A transcription map of the DiGeorge and velo-cardio-facial syndrome minimal 
critical region on 22ql 1 . Hum Mol Genet 5: 789-800. 

Goodrich KH, Holcombe RF. (1995) Genetic localization of the gene for Chediak- 
Higashi syndrome to human chromosome lq and linkage to nidogen. J Investig Med 
43(1), Suppl.l,13A. 

Green EL. (1981) Linkage, recombination and mapping. In "Genetics and Probabilities in 
Animal Breeding Experiments," pp. 77-1 13, Macmillan, New York. 

Grossi CE, Crist WM, Abo T, Velardi A, Cooper MD. (1985) Expression of the Chediak- 
Higashi lysosomal abnormality in human peripheral blood lymphocyte subpopulations. 
Blood 65(4): 837-44. 

Guo H, Sekiguchi M, Shimai K. (1992) Cytoarchitectonic abnormalities in cerebellum 
and hipocampal formation of beige mutant mouse. TokaiJ Exp Clin Med 17(1), 53-61. 

Hamanaka SC, Gilbert CS, White DA, Parmley RT. (1993) Ultrastructural morphology, 
cytochemistry, and morphometry of eosinophil granules in Chediak-Higashi syndrome. 
Am J Pathol 143(2): 618-27. 

Hayakawa H, Kobayashi N, Yata J. (1986) Primary immunodeficiency diseases and 
malignancy in Japan. Jpn J Cancer Res. 77(1), 74-9. 

Henikoff S, Henikoff JG. (1991) Automated assembly of protein blocks for database 
searching. Nucleic Acids Research 19 (23):6565-6572. 

Higashi O. (1954) Congenital gigantism of peroxidase granules. The first case ever 
reported of qualitative abnormality of peroxidase. The Tohoku J Exp Med 59, 315. 



182 



Hillier L, Clark N, Dubuque T, Elliston K, Hawkins M, Holman M, Hultman M, Kucaba 
T, Le M, Lennon G, Marra M, Parsons J, Rifkin L, Rohlfing T, Soares M, Tan F, 
Trevaskis E, Waterson R, Williamson A, Wohldmann P, Wilson R. (1995, 1997) WashU- 
Merck EST Project. 

Hiding H, Scheller RH. (1996) Phosphorylation of synaptic vesicle proteins: modulation 
of the aSNAP interaction with the core complex. Proc Natl Acad Sci 93: 1 1945-49. 

Holcombe RF. (1992) Interleukin-2-induced cytotoxicity of Chediak-Higashi 
lymphocytes. Acta Hematol 87(l-2):45-8. 

Holcombe RF, Jones KL, Stewart RM. (1994) Lysosomal enzyme activities in Chediak- 
Higashi syndrome: evaluation of lymphoblastoid cell lines and review of the literature. 
Immunudeficiency 5 , 1 3 1 - 1 40 . 

Holcombe RF, Stephenson DA, Zweidler A, Stewart RM, Chapman VM, Seidman JG. 
(1991) Linkage of loci associated with two pigment mutations on mouse Chromosome 
13. Genet Res 58, 41-50. 

Holcombe RF, Strauss W, Owen FL, Boxer LA, Warren RW, Conley ME, Ferrara J, 
Leavitt RY, Fauci AS, Taylor BA, Seidman JG. (1987) Relationship of the genes for 
Chediak-Higashi syndrome (beige) and the T-cell receptor y-chain in mouse and man. 
Genomics 1:287-91. 

Holcombe RF, van de Griend R, Ang SL, Bolhuis RL, Seidman JG. (1990) Gamma-delta 
T cells in Chediak-Higashi syndrome. Acta Hematol 83(4): 193-7. 

Ishimura T, Isobe T, Okuyama T, Yamauchi T, Fujisawa H. (1987) Brain 14-3-3 protein 
is an activator protein that activates tryptophan 5-monooxygenase and tyrosine 3- 
monooxygenase in the presence of Ca 2+ , calmodulin-dependent protein kinase II. FEBS 
Lett 219:79-82. 

Ishino M, Ohba T, Inazawa J, Sasaki H, Ariyama Y, Sasaki T. (1997) Identification of an 
Efs isoform that lacks the SH3 domain and chromosomal mapping of human Efs. 
Oncogene 15: 1741-45. 

Isobe T, Hiyane Y, Ishimura T, Okuyama T, Takahashi N, Nakajo S, Nakaya K. (1992) 
Activation of protein kinase C by the 14-3-3 proteins homologous with Exol protein that 
stimulates calcium-dependent exocytosis. FEBS Lett 308:121-24. 

Ito H, Fukuda Y, Murata K, Kimura A. (1983) Transformation of intact yeast cells treated 
with alkali cations. J Bacteriol 153(1):163-168. 



183 



Ito M, Sato A, Tanabe F, Ishida E, Takami Y, Shigeta S. (1989) The thiol proteinase 
inhibitors improve the abnormal rapid down-regulation of protein kinase C and the 
impaired natural killer cell activity in (Chediak-Higashi syndrome) beige mouse. 
Biochem Biophys Res Commun 160: 433-40. 

Ito M, Tanabe F, Takami Y, Sato A, Shigeta S. (1988) Rapid down-regulation of protein 
kinase C in (Chediak-Higashi syndrome) beige mouse by phorbol ester. Biochem Biophys 
Res Commun 153: 648-56. 

Jenkins NA, Justice MA, Gilbert DJ, Chu ML, Copeland NG. (1991) Nidogen/entactin 
(Nid) maps to the proximal end of mouse chromosome 13 linked to beige (bg) and 
identifies a new region of homology between mouse and human chromosomes. Genomics 
9:401-3. 

Justice MJ, Silan CM, Ceci JD, Buchberg AM, Copeland NG, Jenkins NA. (1990) A 
molecular genetic linkage map of mouse chromosome 13 anchored by the beige (bg) and 
satin (sa) loci. Genomics 6:341-351. 

Kaerre K, Klein GO, Kiessling R, Klein G, Roder JC. (1980) Low natural in vivo 
resistance to syngeneic leukaemias in natural killer-deficient mice. Nature 284(5757): 
624-6. 

Kaiser C, Michaelis S, Mitchell A. (1994) Methods in Yeast Genetics, Cold Spring 
Harbor Laboratory Press, New York, p. 209. 

Karim MA, Nagle DL, Kandil HH, Buerger J, Moore KJ, Spritz RA. (1997) Mutations in 
the Chediak-Higashi syndrome gene (CHS1) indicate requirement for the complete 3801 
amino acid CHS protein. Hum Mol Genet 6: 1087-89. 

Kingsmore SF, Barbosa MDFS, Nguyen QA, Ashley JA, Blaydes SM, Tchernev VT, 
Detter CD, Lovett M. (1996b) Physical mapping of the beige critical region on mouse 
chromosome 13. Mamm Genome 7:773-5. 

Kingsmore SF, Barbosa MDFS, Tchernev VT, Detter CD, Lossie AC, Seldin MF, 
Holcmbe RF. (1996a) Positional cloning of the Chediak-Higashi syndrome gene: genetic 
mapping of the beige locus on mouse chromosome 13. J Invest MeJ44(8):454-61. 

Kinugawa N, Ohtani T. (1985) Beneficial effects of high-dose intravenous 
gammaglobulin on the accelerated phase of Chediak-Higashi syndrome. Helv Paediatr 
^cto40(2-3):169-172. 

Knight SP, Richardson MM, Osmand AP, Stakkestad A, Potter NT. (1997) Expression 
and distribution of the dentatorubral-pallidoluysian atrophy gene product (atrophin 
1/drplap) in neuronal and non-neuronal tissues. J Neurol Sci 146: 19-26. 



184 



Koffer A, Tatham PER, Gomperts BD. (1990) Changes in the state of actin during the 
exocytotic reaction in mast cells. J Cell Biol 111:91 9-27. 

Komada M, Kitamura N. (1994) Regulatory role of major tyrosine autophosphorylation 
site of kinase domain of c-Met receptor (scatter factor/hepatocyte growth factor receptor. 
J Biol Chem 269: 16131-36. 

Komada M, Kitamura N. (1995) Growth factor-induced tyrosine phosphorylation of Hrs, 
a novel 1 1 5-kilodalton protein with a structurally conserved putative zinc finger domain. 
Mol Cell Biol 15: 6213-21. 

Komada M, Masaki R, Yamamoto A, Kitamura N. (1997) Hrs, a tyrosine kinase substrate 
with conserved double zinc finger domain, is localized to the cytoplasmic surface of early 
endosomes. J Biol Chem 272: 20538-44. 

Kondo N, ShimozawaN, Asano J, Imamura A, Orii T. (1994) Chediak-Higashi syndrome 
with cerebellar cortical atrophy detected by MRI. Clin Genet 46(6), 439-40. 

Kreimer DJ, Khotimchenko YS. (1995) Cytoplasm calcium-binding proteins of germ 
cellsand embryos of the sea urchin. Comp Biochem Physiol 1 10A: 95-105. 

Kubo A, Sasada M, Nishimura T, Moriguchi T, Kakita T, Yamamoto K, Uchino H. 
(1987) Oxygen radical generation by polymorphonuclear leukocytes of beige mice. Clin 
Exp Immunol 70(3):658-63. 

Lambright DG, Sondek J, Bohm A, Skiba NP, Hamm HE, Sigler PB. (1996) The 2 A 
crystal structure of a heterotrimeric G protein. Nature 379:31 1-19. 

Lane PW. (1971) Xt-bg-cr linkage. Mouse News Lett 45:29. 

Laskey RA, Goerlich D, Madine MA, Makkerh JPS, Romanowski P. (1996) Regulatory 
roles of the nuclear envelope. Exp Cell Res 229: 204-1 1 . 

Lockman LA, Kennedy WR, White JG. (1967) The Chediak-Higashi 
syndrome:electrophysiological and electron microscopic observations on the peripheral 
neuropathy. J Pediatr 70, 942-51. 

Lyon MF, Meredith R. (1969) Muted, a new mutant affecting coat colour and otoliths of 
the mouse, and its position in linkage group XIV. Genet Res 14:163-66. 

Mahoney KH, Morse SS, Morahan PS. (1980) Macrophage functions in beige 
(Chediak-Higashi syndrome) mice. Cancer Res 40(1 1): 3934-9. 



185 



Margolis RL, Li SH, Young WS, Wagster MV, Stine OC, Kidwai AS, Ashworth RG, 
Ross CA. (1996) DRPLA gene (atrophin 1) sequence and mRNA expression in human 
brain. Mol Brain Res 36: 219-26. 

Marklund U, Larsson N, Gradin HM, Brattsand G, Gullberg M. (1996) Oncoprotein 18 is 
a phosphorylation-responsive regulator of microtubule dynamics. EMBO J 15:5290-98. 

Martin H, Rostas J, Patel Y, Aitken A. (1994) Analysis of subcellular distribution of 14- 
3-3 isoforms in rat brain using specific antibodies. J Neurochem 63:2259-65. 

Matsuda M, Okabe T, Sugimoto N, Senda T, Fujita H. (1994) Tetanus toxin and 
Clostridium perfringens enterotoxin as tools for the study of exocytosis. Ann N Y Acad 
Sci 710: 94-106. 

Matteoni R, Kreis TE. (1987) Translocation and clustering of endosomes and lysosomes 
depends on microtubules. J Cell Biol 105:1253-65. 

Menard M, Meyers KM. (1988) Storage pool deficiency in cattle with Chediak-Higashi 
syndrome results from an absence of dense granule precursors in their megakaryocytes. 
Blood 72(5): 1726-34. 

Merino F, Klein GO, Henle W, Ramirez-Duque P, Forsgren M, Amesty C. (1983) 
Elevated antibody titers to Epstein-Barr virus and low natural killer cell activity in 
patients with Chediak-Higashi syndrome. Clin Immunol Immunopathol 27(3):326-39. 

Meyers KM, Seachord CL, Benson K, Fukami M, Holmsen H. (1983) Serotonin 
accumulation in granules of storage pool-deficient platelets of Chediak-Higashi cattle. 
Am J Physiol 245(1): HI 50-8. 

Meyers KM, Stevens DR, Padgett GA. (1974) A platelet serotonin anomaly in the 
Chediak-Higashi syndrome. Res Commun Chem Pathol Pharmacol. 7(2), 375-80. 

Misra VP, King RH, Harding AE, Muddle JR, Thomas PK. (1991) Peripheral neuropathy 
in the Chediak-Higashi syndrome. Acta Neuropathol Berl. 81(3), 354-8. 

Mitchell PJ, Tjian R. (1989) Transcriptional regulation in mammalian cells by sequence- 
specific DNA binding proteins. Science 245 (4916): 371-8. 

Morrison D. (1994) 14-3-3: modulators of signaling proteins? Science 266: 56-7. 

Morrison D. (1995) Mechanisms regulating Rafl activity in signal transduction 
pathways. Mol Reprod Dev 42: 507-14. 

Moskowitz N, Schook W, Lisanto M, Hua E, Puszkin S. (1982) Calmodulin affinity for 
brain coated vesicle protein. J Neurochem 38: 1742. 



186 



Murphy ED, Roths JB. (1978) Purkinje cell degeneration, a late effect of beige mutations 
in mice. Jackson Lab Ann Rep 49, 108-9. 

Myers SM, Eng C, Ponder BAJ, Mulligan LM. (1995) Characterization of RET proto- 
oncogene 3' splicing variants and polyadenylation sites: a novel C-terminus for RET. 
Oncogene 11:2039-45. 

Nagase T, Seki N, Ishikawa K, Tanaka A, Nomura N. (1996) Prediction of the coding 
sequences of unidentified human genes. V. The coding sequences of 40 new genes 
(KIAA0161 - KIAA0200) deduced by analysis of cDNA clones from human cell line 
KG-\.DNARes. 3(1): 17-24. 

Nagle DL, Karim MA, Woolf EA, Holmgren L, Bork P, Misumi DJ, McGrail SH, 
Dussault BJ, Perou CM, Boissy RE, Duyk GM, Spritz RA, Moore KJ. (1996) 
Identification and mutation analysis of the complete gene for Chediak-Higashi syndrome. 
Nat Genet 14:307-11. 

Nakai K, Kanehisa M. (1992) A knowledge base for predicting protein localization sites 
in eukaryotic cells. Genomics 14:897-911. 

Novak EK, McGarry MP, Swank RT. (1985) Correction of symptoms of platelet storage 
pool deficiency in animal models for Chediak-Higashi syndrome and Hermansky-Pudlak 
syndrome. Blood 66(5): 1196-201. 

Oka JA, Weigel PH. (1983) Microtubule-depolymerizing agents inhibit asialo- 
orosomucoid delivery to lysosomes but not its endocytosis or degradation in isolated rat 
hepatocytes. Biochim Biophys Acta 763:368-76. 

Oliver JM, Zurier RB, Berlin RD. (1975) Concavalin A cap formation on 
polymorphonuclear leukocytes of normal and beige (Chediak-Higashi) mice. Nature 
253:471-73. 

Olsen DR, Nagayoshi T, Fazio M, Mattei M-G, Passage E, Weil D, Timpl R, Chu M-L, 
Uitto J. (1989) Human nidogen: cDNA cloning, cellular expression, and mapping of the 
gene to chromosome lq43. Amer J Hum Genet 44:876-885. 

Owen FL, Taylor BA, Zweidler A, Seidman JG. (1986) The murine y-chain of the T cell 
receptor is closely linked to a spermatocyte specific histone gene and the beige coat color 
locus on chromosome 13. J Immunology 137:1044-46. 

Peifer M, Berg S, Reynolds A. (1994) A repeating amino-acid motif shared by proteins 
with diverse cellular functions. Cell 76: 789-91. 



187 



Penner JD, Prieur DJ. (1987) Interspecific genetic complementation analysis with 
fibroblasts from humans and four species of animals with Chediak-Higashi syndrome. 
Am J Med Genet. 28(2), 455-70. 

Perin MS, Brose N, Jahn R, Suedhof TC. (1991) J Biol Chem 266:623-29. 

Perou C, Justice MJ, Pryor RJ, Kaplan J. (1996a) Complementation of the beige mutation 
in cultured cells by episomally replicating murine yeast artificial chromosomes. Proc 
Natl Acad Sci USA 93: 5905-09. 

Perou CM, Kaplan J. (1993) Chediak-Higashi syndrome is not due to a defect in 
microtubule-based lysosomal mobility. J Cell Sci. 106 (Pt 1), 99-107. 

Perou CM, Moore KJ, Nagle DL, Misumi DJ, Woolf EA, McGrail SH, Holmgren L, 
Brody BJD, Monroe CA, Duyk GM, Pryor RJ, Li L, Justice M, Kaplan J. (1996b) 
Identification of the murine beige gene by YAC complementation and positional cloning. 
Nature Genetics 13, 303-308. 

Perrin D, Moeller K, Hanke K, Soeling HD. (1992) cAMP and Ca 2+ mediated secretion in 
parotid acinar cells is associated with reversible changes in the organization of the 
cytoskeleton.yCe//5/o/ 116: 127-34. 

Pettit RE, Berdal KG. (1984) Chediak-Higashi syndrome. Neurologic appearance. Arch 
Neurol 41(9), 1001-2. 

Pezeshkpour G, Kurent JS, Krarup C, Buchthal F, Fauci AS. (1986) Peripheral 
neuropathy in Chediak-Higashi syndrome. J Neuropathol Exp Neurol 45: 353. 

Pflumio F, Fonteneau P, Loor F. (1990) Impaired antibody response of C57BL/6 beige 
mutant mice to a thymus-independent type 2 antigen. Immunol Lett 23(4): 269-74. 

Phillips LL, Kaplan HA, Padgett GA, Gorham JR. (1967) Comparative studies on the 
Chediak-Higashi syndrome: coagulation and fibrinolytic mechanisms of mink and cattle. 
Am J Vet Clin Path 1: 1-6. 

Pratt HL, Carroll RC, Jones JB, Lothrop CD Jr. (1991) Platelet aggregation, storage pool 
deficiency, and protein phosphorylation in mice with Chediak-Higashi syndrome. Am J 
Vet Res 52(6): 945-50. 

Ramirez-Duque P, Arends T, Merino F. (1982) Chediak-Higashi syndrome: description 
of a cluster in a Venezuelan- Andean isolated region. J Medicine, 13 (5-6), 431-51. 

Rendu F, Breton-Gorius J, Lebret M, Klebanoff C, Buriot D, Griscelli C, Levy-Toledano 
S, Caen JP. (1983) Evidence that abnormal platelet functions in human Chediak-Higashi 
syndrome are the result of a lack of dense bodies. Am J Pathol 1 1 1(3): 307-14. 



188 



Reuther GW, Fu H, Cripe LD, Collier RJ, Pendergast AM. (1994) Association of the 
protein kinases c-Bcr and Bcr-Abl with proteins of the 14-3-3 family. Science 266: 129- 
133. 

Roder J, Duwe A. (1979) The beige mutation in the mouse selectively impairs natural 
killer cell function. Nature 278(5703): 451-3. 

Roder JC, Haliotis T, Laing L, Kozbor D, Rubin P, Pross H, Boxer LA, White JG, Fauci 
AS, Mostowski H, Matheson DS. (1982) Further studies of natural killer cell function in 
Chediak-Higashi patients. Immunology 46(3): 555-60. 

Roder JC, Lohmann-Matthes ML, Domzig W, Wigzell H. (1979) The beige mutation in 
the mouse. II. Selectivity of the natural killer (NK) cell defect. J Immunol 123(5): 
2174-81. 

Roder JC, Todd RF, Rubin P, Haliotis T, Helfand SL, Werkmeister J, Pross HF, Boxer 
LA, Schlossman SF, Fauci AS. (1983) The Chediak-Higashi gene in humans. III. Studies 
on the mechanisms of NK impairment. Clin Exp Immunol 51(2): 359-68. 

Roep BO, Arden SD, de Vries RRP, Hutton JC. (1990) T-cell clones from a type 1 
diabetes patient respond to insulin secretory granule proteins. Nature 345: 632-34. 

Ross CA, Becher MW, Colomer V, Engelender S, Wood JD, Sharp AH. (1997) 
Huntington's disease and dentatorubral-pallidoluysian atrophy: proteins, pathogenesis 
and pathology. Brain Pathol 7: 1003-16. 

Roth D, Burgoyne RD. (1995) Stimulation of catecholamine secretion from adrenal 
chromaffin cells by 14-3-3 proteins is due to reorganisation of the cortical actin network. 
FEBS Lett 374: 77-81. 

Roth D, Morgan A, Martin H, Jones D, Martens GJM, Aitken A, Burgoyne RD. (1994) 
Characterization of 14-3-3 proteins in adrenal chromaffin cells and demonstration of 
isoform-specific phospholipid binding. Biochem J 301: 305-10. 

Sambrook J, Fritsch EF, Maniatis T. (1989) In "Molecular cloning: a laboratory manual", 
Vol. 2, p. 9.16-9.23, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

Sato A. (1955) Chediak and Higashi's disease: probable identity of "a new leucocytal 
anomaly (Chediak)" and "congenital gigantism of peroxidase granules (Higashi)". The 
Tohoku J Exp Med, 61,201. 

Sato A, Tanabe F, Ito M, Ishida E, Shigeta S. (1990) Thiol proteinase inhibitors reverse 
the increased protein kinase C down-regulation and concavalin A cap formation in 



189 



polymorphonuclear leukocytes from Chediak-Higashi syndrome (beige) mouse. J Leukoc 
Biol 48: 377-81. 

Schiebler W, Jahn R, Doucet JP, Rothlein J, Greengard P. (1986) Characterization of 
synapsin I binding to small synaptic vesicles. J Biol Chem 261: 8383-90. 

Sherman F. (1991) Getting started with yeast. Methods Enzymol 194: 3-21. 

Shinozaki K, Maruyama K, Kume H, Kuzume H, Obata K. (1997) A novel brain gene, 
norbin, induced by treatment of tetraethylammonium in rat hippocampal slice and 
accompanied with neurite-outgrowth in neuro 2a cells. Biochem Biophys Res Commun, 
240 (3): 766-71. 

Sjaastad OV, Blom AK, Stormorken H, Nes N. (1990) Adenine nucleotides, serotonin, 
and aggregation properties of blue foxes (Alopex lagopus) with the Chediak-Higashi 
syndrome. Am J Med Genet 35(3): 373-8. 

Sobel A. (1991) Stathmin: a relay phosphoprotein for multiple signal transduction? 
Trends Biochem Sci 16:301-05. 

St. Amand W, Cupp MB. (1958) New linkage. Mouse News Lett 19:38. 

Steinbrinck W. (1948) Ueber eine neue Granulationsanomalie der Leukocyten. Deutsch 
Archiv Klin Med, 193, 577. 

Sugimoto Y, Kusakabe T, Kai T. (1995) Analysis of the in vitro translation product of a 
novel-type Drosophila melanogaster aldolase mRNA in which two carboxy-terminal 
exons remain unspliced. Arch Biochem Biophys 323: 361-66. 

Sung JH, Meyers JP, Stadlan EM, Cowen D, Wolf A. (1969) Neuropathological changes 
in Chediak-Higashi disease. J Neuropathol Exp Neurol 28, 86-1 18. 

Swanson JA, Bushnell A, Silverstein SC. (1987) Tubular lysosome morphology and 
distribution within macrophages depend upon the integrity of cytoplasmic microtubules. 
Proc Natl Acad Sci USA 84:1921-25. 

Swanson JA, Locke A, Ansel P, Hollenbeck PJ. (1992) Radial movement of lysosomes 
along microtubules in permealized macrophages. J Cell Sci 103:210-9. 

Sygiyama T, Nishio Y, Kishimoto T, Akira S. (1996) Identification of alternative splicing 
form of Stat2. FEBS Lett 381:191-94. 

Talmage JE, Meyers KM, Prieur DJ, Starkey JR. (1980) Role of NK cells in tumour 
growth and metastasis in beige mice. Nature 284:622-4. 



190 



Targan SR, Oseas R. (1983) The "lazy" NK cells of Chediak-Higashi syndrome. J 
Immunol 130(6): 2671-4. 

Tchernev VT, Barbosa MDFS, Detter JC, Patel TD, Achey K, Wakeland EK, 
Gueorguieva RV, Yang MCK, Gossler A, Kingsmore SF (1997). Genetic mapping of 20 
novel expressed sequence tags from midgestation mouse embryos suggests chromosomal 
clustering. Genomics 40, 170-174. 

Toker A, Ellis CA, Sellers LA, Aitken A. (1990) Protein kinase C inhibitor proteins. 
Purification from sheep brain and sequence similarity to lipocortin and 14-3-3 protein. 
EurJBiochem 191: 421-29. 

Toker A, Sellers LA, Amess B, Patel Y, Harris A, Aitken A. Multiple isoforms of a 
protein kinase C inhibitor (KCIP- 1/1 4-3-3) from sheep brain. Amino acid sequence of 
phosphorylated forms. (1992) EurJBiochem 206: 453-461. 

Trapp T, Holsboer F. (1996) Nuclear orphan receptor as a repressor of glucocorticoid 
receptor transcriptional activity. J Biol Chem 17: 9879-82. 

Trifaro JM, Vitale ML, Del Castillo AR. (1992) Cytoskeleton and molecular mechanisms 
in neurotransmitter release by neurosecretory cells. Europ J Pharmacol 225: 83-104. 

Uyama E, Hirano T, Ito K, Nakashima H, Sugimoto M, Naito M, Uchino M, Ando M. 
(1994) Adult Chediak-Higashi syndrome presenting as parkinsonism and dementia. Acta 
Neurol Scand 89(3), 1 75-83 . 

Van De Wetering M, Castrop J, Koriinev V, Clevers H. (1996) Extensive alternative 
splicing and dual promoter usage generate Tcf-1 protein isoforms with differential 
transcription control properties. Mol Cell Biol 16:745-752. 

Virelizier JL, Griscelli C. (1980) Interferon administration as an immunoregulatory and 
antimicrobial treatment in children with defective interferon secretion. Primary 
immunodeficiencies. Elsevier/North Holland Biomedical Press, 1980. Pp. 473-84. 

Wallace JC, Henikoff S. (1992) PATMAT: a searching and extraction program for 
sequence, pattern and block queries and databases. Comput Appl Biosci 8(3):249-254. 

Watkins DT, Cooperstein SJ. (1983) Role of calcium and calmodulin in the interaction 
between islet cell secretion granules and plasma membranes. Endocrinology 112: 766. 

Wiese S, Murphy DB, Schlung A, Burfeind P, Schmundt D, Schnuelle V, Mattei MG, 
Thies U. (1995) The genes for human brain factor 1 and 2, members of the fork head gene 
family, are clustered on chromosome 14q. Biochim Biophys Acta 1262: 105-112. 



191 



Willingham MC, Spicer SS, Vincent RA Jr. (1981) The origin and fate of large dense 
bodies in beige mouse fibroblasts. Lysosomal fusion and exocytosis. Exp Cell Res 
136(1), 157-68. 

Windhorst DB, Zelickson AS, Good RA. (1966) Chediak-Higashi syndrome: hereditary 
gigantism of cytoplasmic organelles. Science, 151, 81-83. 

Windhorst DB, Zelickson AS, Good RA. (1968) A human pigmentary dilution based on a 
heritable subcellular structural defect~the Chediak-Higashi syndrome. J Invest Dermatol. 
50(1), 9-18. 

Yakel JL. (1997) Calcineurin regulation of synaptic function: from ion channels to 
transmitter release and gene transcription. Trends Pharmacol Sci 18: 124-34. 

Yamashita T, Wu N, Kupfer G, Corless C, Joenje H, Grompe M, D' Andrea AD. (1996) 
Clinical variability of Fanconi anemia (Type C) results from expression of an amino 
terminal truncated Fanconi anemia complementation group C polypeptide with partial 
activity. Blood 87:4424-32. 

Yang N, Shigeta H, Shi H, Teng CT. (1996) Estrogen-related receptor, hERRl, modulates 
estrogen receptor-mediated response of human lactoferrin gene promoter. J Biol Chem 
271:5795-804. 

Yazawa I, Nukina N, Hashida H, Goto J, Yamada M, Kanazawa I. (1995) Abnormal gene 
product identified in hereditary dentatorubral-pallidoluysian atrophy (DRPLA) brain. Nat 
Genet 10: 99-103. 

Yegin O, Sanal O, Yeralan O, Gurgey A, Berkel AI. (1983) Defective lymphocyte 
locomotion in Chediak-Higashi syndrome. Am J Dis Child 137(8):771-3. 

Zhao H, Boissy YL, Zalfa AM, King RA, Nordlund JJ, Boissy RE. (1994) On the 
analysis of the pathophysiology of Chediak-Higashi syndrome. Defects expressed by 
cultured melanocytes. Lab Investigation, 71, 25-34. 

Zhao W, Manlley JL. (1996) Complex alternative mRNA processing generates an 
unexpected diversity of poly(A) polymerase forms. Mol Cell Biol 16:2378-2386. 

Zupan LA, Steffens DL, Berry ML, Gross RW. (1992) J Biol Chem 267:8707-10. 



BIOGRAPHICAL SKETCH 



Velizar T. Tchernev was born in Sofia, Bulgaria. In 1987, he graduated from high 
school with extensive study of the German language in Sofia. Velizar Tchernev received 
his Doctor of Medicine degree from the Higher Medical Institute-Sofia, Bulgaria, in 

1993. He also obtained diplomas for Translator of Medical Literature from German 
(1990) and from English (1991) languages from the Higher Medical Institute-Sofia. In 

1994, Velizar Tchernev entered the Ph.D. program in Immunology and Molecular 
Pathology at the Department of Pathology and Laboratory Medicine, College of 
Medicine, University of Florida. 



192 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Stephen F. Kingsmore, Cngjr 
Assistant Professor of Pathology, 
Immunology and Laboratory Medicine 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 



^{toi&uif- £* CVq^<^ _ 



Margaret R. Wallace 

Associate Professor of Pediatrics 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 



£L-1 £Jl(7 



Eric S. Sobel 

Associate Professor of Medicine 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 



,/LUvmk 

Michael R. Bubb 

Assistant Professor of Medicine 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 



Edward K. Wakeland 
Professor of Pathology, Immunology and 
Laboratory Medicine 



This dissertation was submitted to the Graduate Faculty of the College of 
Medicine and to the Graduate School and was accepted as partial fulfillment of the 
requirements for the degree of Doctor of Philosophy. 



December 1998 



Dean, College of Medicine 




Dean, Tjraduate Scho 



UNIVERSITY OF FLORIDA 



3 1262 08555 3021